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Sample records for avian adeno-associated virus

  1. The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

    Institute of Scientific and Technical Information of China (English)

    WANG An-ping; SUN Huai-chang; WANG Jian-ye; WANG Yong-juan; YUAN Wei-feng

    2007-01-01

    To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

  2. Adeno-associated virus for cystic fibrosis gene therapy

    Directory of Open Access Journals (Sweden)

    S.V. Martini

    2011-11-01

    Full Text Available Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR. The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR to the affected organ (lung. Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.

  3. Adeno-associated virus: from defective virus to effective vector

    Directory of Open Access Journals (Sweden)

    Gonçalves Manuel AFV

    2005-05-01

    Full Text Available Abstract The initial discovery of adeno-associated virus (AAV mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.

  4. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  5. Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

    OpenAIRE

    Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2008-01-01

    Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution.

  6. Evolutionary Relationships among Parvoviruses: Virus-Host Coevolution among Autonomous Primate Parvoviruses and Links between Adeno-Associated and Avian Parvoviruses

    OpenAIRE

    Lukashov, Vladimir V.; Goudsmit, Jaap

    2001-01-01

    The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family Parvoviridae. Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary ...

  7. Structure of neurotropic adeno-associated virus AAVrh.8.

    Science.gov (United States)

    Halder, Sujata; Van Vliet, Kim; Smith, J Kennon; Duong, Thao Thi Phuong; McKenna, Robert; Wilson, James M; Agbandje-McKenna, Mavis

    2015-10-01

    Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5 Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ∼ 84%, ∼ 91%, and ∼ 87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies and human donor serum directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment. PMID:26334681

  8. Adeno-associated virus rep protein synthesis during productive infection

    Energy Technology Data Exchange (ETDEWEB)

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  9. Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

    OpenAIRE

    Lerch, Thomas F.; Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2009-01-01

    Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning.

  10. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

    OpenAIRE

    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su; Han, Su-Cheol; Lee, C. Justin

    2016-01-01

    Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey br...

  11. Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

    OpenAIRE

    Qiao, Chunping; Li, Juan; Skold, Anna; Zhang, Xudong; Xiao, Xiao

    2002-01-01

    The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection ...

  12. Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

    Energy Technology Data Exchange (ETDEWEB)

    Yakobson, B.; Hrynko, T.A.; Peak, M.J.; Winocour, E.

    1989-03-01

    Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting a requirement for de novo protein synthesis.

  13. Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

    International Nuclear Information System (INIS)

    Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning. Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication

  14. Adeno-Associated Virus Serotype-9 Microdystrophin Gene Therapy Ameliorates Electrocardiographic Abnormalities in mdx Mice

    OpenAIRE

    Bostick, Brian; Yue, Yongping; Lai, Yi; Long, Chun; Li, Dejia; Duan, Dongsheng

    2008-01-01

    Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy norm...

  15. Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

    OpenAIRE

    Yang Lin; Xiao Xiao

    2013-01-01

    Abstract Adeno-associated virus (AAV) is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolutio...

  16. Rapid, simple and versatile manufacturing of recombinant adeno-associated virus vectors at scale

    OpenAIRE

    Lock, Martin; Alvira, Mauricio; Vandenberghe, Luk H.; Samanta, Arabinda; Toelen, Jaan; Debyser, Zeger; Wilson, James M

    2010-01-01

    Adeno-associated virus vector manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. While scalable systems based upon AAV-adenovirus, -herpesvirus and -baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate scale pre-clinical studies in large animals where several combinations of serotype and genome may need to be tested. Recently we observed that d...

  17. Adeno-Associated Virus-Mediated Cancer Gene Therapy: Current Status

    OpenAIRE

    Luo, Jingfeng; Luo, Yuxuan; Sun, Jihong; Zhou, Yurong; Zhang, Yajing; Yang, Xiaoming

    2014-01-01

    Gene therapy is one of the frontiers of modern medicine. Adeno-associated virus (AAV)-mediated gene therapy is becoming a promising approach to treat a variety of diseases and cancers. AAV-mediated cancer gene therapies have rapidly advanced due to their superiority to other gene-carrying vectors, such as the lack of pathogenicity, the ability to transfect both dividing and non-dividing cells, low host immune response, and long-term expression. This article reviews and provides up to date kno...

  18. Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction.

    Science.gov (United States)

    Xiao, Ping-Jie; Mitchell, Angela M; Huang, Lu; Li, Chengwen; Samulski, R Jude

    2016-04-01

    Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

  19. Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

    International Nuclear Information System (INIS)

    Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution. Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes. Towards the determination of a structure for AAV type 6, this paper reports the high-yield production, purification, crystallization and preliminary diffraction studies of infectious AAV-6 virions. The crystals diffracted to 3.2 Å resolution using synchrotron radiation. The most promising crystal form belonged to space group R3 and appeared to be suitable for initial structure determination

  20. The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification.

    Science.gov (United States)

    Heilbronn, R; Bürkle, A; Stephan, S; zur Hausen, H

    1990-01-01

    Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification. Images PMID:2159559

  1. Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

    International Nuclear Information System (INIS)

    The production, purification, crystallization and preliminary X-ray crystallographic analysis of adeno-associated virus serotype 8 is reported. Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is ∼80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 Å resolution using synchrotron radiation and belong to the hexagonal space group P6322, with unit-cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit

  2. Adeno-associated virus sensitizes HeLa cell tumors to gamma rays.

    Science.gov (United States)

    Walz, C; Schlehofer, J R; Flentje, M; Rudat, V; zur Hausen, H

    1992-01-01

    Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment. Images PMID:1323717

  3. Systemic gene delivery to the central nervous system using Adeno-associated virus

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    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  4. A novel and highly efficient production system for recombinant adeno-associated virus vector

    Institute of Scientific and Technical Information of China (English)

    WU; Zhijian(伍志坚); WU; Xiaobing(吴小兵); CAO; Hui(曹晖); DONG; Xiaoyan(董小岩); WANG; Hong(王宏); HOU; Yunde(侯云德)

    2002-01-01

    Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

  5. Size does matter: overcoming the adeno-associated virus packaging limit

    Directory of Open Access Journals (Sweden)

    Flotte Terence R

    2000-07-01

    Full Text Available Abstract Recombinant adeno-associated virus (rAAV vectors mediate long-term gene transfer without any known toxicity. The primary limitation of rAAV has been the small size of the virion (20 nm, which only permits the packaging of 4.7 kilobases (kb of exogenous DNA, including the promoter, the polyadenylation signal and any other enhancer elements that might be desired. Two recent reports (D Duan et al: Nat Med 2000, 6:595-598; Z Yan et al: Proc Natl Acad Sci USA 2000, 97:6716-6721 have exploited a unique feature of rAAV genomes, their ability to link together in doublets or strings, to bypass this size limitation. This technology could improve the chances for successful gene therapy of diseases like cystic fibrosis or Duchenne muscular dystrophy that lead to significant pulmonary morbidity.

  6. Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

    Science.gov (United States)

    Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

    2015-03-01

    Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

  7. Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Chun Zhang; Shibo Tang; Yan Luo; Xiaoling Liang; Jing Ma; Shaofen Lin

    2003-01-01

    Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

  8. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

    OpenAIRE

    Samulski, R J; Chang, L S; Shenk, T

    1989-01-01

    A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expre...

  9. Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

    OpenAIRE

    Duan, Dongsheng; Yan, Ziying; Yue, Yongping; Ding, Wei; Engelhardt, John F.

    2001-01-01

    Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improv...

  10. Directed evolution of novel adeno-associated viruses for therapeutic gene delivery.

    Science.gov (United States)

    Bartel, M A; Weinstein, J R; Schaffer, D V

    2012-06-01

    Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties. PMID:22402323

  11. Functional analysis of the putative integrin recognition motif on adeno-associated virus 9.

    Science.gov (United States)

    Shen, Shen; Berry, Garrett E; Castellanos Rivera, Ruth M; Cheung, Roland Y; Troupes, Andrew N; Brown, Sarah M; Kafri, Tal; Asokan, Aravind

    2015-01-16

    Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system. PMID:25404742

  12. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy

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    Hangjun Ruan

    2001-01-01

    Full Text Available The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE from the erythropoietin gene (Epo, which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1. Under anoxic conditions, nine copies of HIRE (9XHRE yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.

  13. Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

    Directory of Open Access Journals (Sweden)

    Yang Lin

    2013-02-01

    Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

  14. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

    Energy Technology Data Exchange (ETDEWEB)

    McCraw, Dustin M. [Department of Biochemistry and Molecular Biology, School of Medicine, Mail code L224, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098 (United States); O& #x27; Donnell, Jason K. [Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380 (United States); Taylor, Kenneth A. [Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295 (United States); Stagg, Scott M. [Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380 (United States); Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306 (United States); Chapman, Michael S., E-mail: chapmami@ohsu.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Mail code L224, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098 (United States)

    2012-09-15

    The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

  15. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  16. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

    Science.gov (United States)

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy. PMID:26414293

  17. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy1

    Science.gov (United States)

    Ruan, Hangjun; Su, Hua; Hu, Lily; Lamborn, Kathleen R; Kan, YW; Deen, Dennis F

    2001-01-01

    Abstract The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen) produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE) from the erythropoietin gene (Epo), which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1). Under anoxic conditions, nine copies of HRE (9XHRE) yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV) in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy. PMID:11494119

  18. Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy.

    Science.gov (United States)

    Nguyen, J T; Wu, P; Clouse, M E; Hlatky, L; Terwilliger, E F

    1998-12-15

    Antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial targeting, an absence of drug resistance. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adeno-associated virus (rAAV) vectors that carry genes coding for angiostatin, endostatin, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines tested. Transduction with an rAAV-encoding antisense VEGF mRNA inhibited the production of endogenous tumor cell VEGF. Conditioned media from cells transduced with this rAAV or with rAAV-expressing endostatin or angiostatin inhibited capillary endothelial cell proliferation in vitro. Antiangiogenic rAAVs may offer a novel gene therapy approach to undermining tumor neovascularization and cancer progression. PMID:9865720

  19. ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

  20. Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

    OpenAIRE

    Lee, Hannah H.; O'Malley, Michael J.; Friel, Nicole A.; Payne, Karin A.; Qiao, Chunping; Xiao, Xiao(Institute for Strings, Cosmology and Astroparticle Physics (ISCAP) and Physics Department, Columbia University, 538 West 120th Street, New York, NY, 10027 U.S.A.); Chu, Constance R.

    2013-01-01

    A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra...

  1. Recombinant adeno-associated viruses (rAAV2) facilitate the intraperitoneal gene delivery to cancer cells

    OpenAIRE

    Malecki, Maciej; PROCZKA, ROBERT; Chorostowska-Wynimko, Joanna; Swoboda, Paweł; DELBANI, ANNA; Pachecka, Jan

    2010-01-01

    Peritoneal dissemination of cancer cells is characteristic of advanced stages of ovarian, breast and lung cancers, and is associated with poor patient survival. The presence of cancer cells in effusions complicates treatment protocols, while cell eradication is seriously limited. One of the novel options available is cancer gene therapy with recombinant adeno-associated viruses. This combination represents the most promising gene delivery vehicles to neoplasmatic cells within serosal cavities...

  2. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

    OpenAIRE

    Wang, Bing; Li, Juan; Xiao, Xiao

    2000-01-01

    Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too sm...

  3. Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

    Directory of Open Access Journals (Sweden)

    Daniel J. Schuster

    2014-08-01

    Full Text Available We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5. Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated astrocytes and neurons. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.

  4. Developing immunologically inert adeno-associated virus (AAV) vectors for gene therapy: possibilities and limitations.

    Science.gov (United States)

    Selot, Ruchita S; Hareendran, Sangeetha; Jayandharan, Giridhara R

    2014-01-01

    Gene therapy has become a clinical reality as demonstrated by remarkable benefits seen in Phase I/II clinical trials for hemophilia B, lipoprotein lipase deficiency and Leber's congenital amarousis. The choice of, and the improved understanding in vector characteristics have contributed significantly to this success. The adeno-associated virus (AAV) vectors used in these trials have been long known to be relatively safe and efficacious. However, certain factors, most notably host immunity to the vector, prevent their widespread use. In patients who have pre-existing antibodies to AAV, these vectors will be rapidly cleared. Administration of a relatively high initial dose of vector to achieve and sustain a higher margin of therapeutic benefit is limited by concerns of vector dose-dependent T cell response. Frequent vector administration necessitated by the non-integrating nature of the virus is difficult due to the variable, yet significant host immunological memory. Thus generation of AAV vectors that are immunologically inert is pivotal for the long-term success with this promising vector system. Several strategies, that aim targeted disruption of antigenic sites or those that chemically modify the vectors have been proposed for host immune evasion. While these approaches have been successful in the pre-clinical model systems, this continues to be a field of intense experimentation and constant improvisation due to limited information available on vector immunology or data from human studies. This review forms a comprehensive report on current strategies available to generate immunologically inert AAV vectors and their potential in mediating longterm gene transfer. PMID:24678652

  5. Establishment of a recombinant adeno-associated virus expressing hVEGF165

    Institute of Scientific and Technical Information of China (English)

    Xianghui Huang; Zhibin Shi; Xiaoqian Dang; Chen Zhang; Pengbo Yu; Kunzheng Wang

    2008-01-01

    BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion

  6. Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids.

    Science.gov (United States)

    Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

    2016-02-01

    Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

  7. Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids†

    Science.gov (United States)

    Kern, A.; Schmidt, K.; Leder, C.; Müller, O. J.; Wobus, C. E.; Bettinger, K.; Von der Lieth, C. W.; King, J. A.; Kleinschmidt, J. A.

    2003-01-01

    Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary. PMID:14512555

  8. Structural characterization of the dual glycan binding adeno-associated virus serotype 6.

    Science.gov (United States)

    Ng, Robert; Govindasamy, Lakshmanan; Gurda, Brittney L; McKenna, Robert; Kozyreva, Olga G; Samulski, R Jude; Parent, Kristin N; Baker, Timothy S; Agbandje-McKenna, Mavis

    2010-12-01

    The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization. PMID:20861247

  9. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  10. Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway▿

    OpenAIRE

    Daya, Shyam; Cortez, Nenita; Berns, Kenneth I.

    2009-01-01

    Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two prot...

  11. Impact of Pre-Existing Immunity on Gene Transfer to Nonhuman Primate Liver with Adeno-Associated Virus 8 Vectors

    OpenAIRE

    Wang, Lili; Calcedo, Roberto; Bell, Peter; Lin, Jianping; Grant, Rebecca L.; Siegel, Don L.; James M Wilson

    2011-01-01

    Vectors based on the primate-derived adeno-associated virus serotype 8 (AAV8) are being evaluated in preclinical and clinical models. Natural infections with related AAVs activate memory B cells that produce antibodies capable of modulating the efficacy and safety of the vector. We have evaluated the biology of AAV8 gene transfer in macaque liver, with a focus on assessing the impact of pre-existing humoral immunity. Twenty-one macaques with various levels of AAV neutralizing antibody (NAb) w...

  12. Adeno-Associated Virus-Mediated Microdystrophin Expression Protects Young mdx Muscle from Contraction-Induced Injury

    OpenAIRE

    LIU, MINGJU; Yue, Yongping; Harper, Scott Q.; Grange, Robert W.; Jeffrey S. Chamberlain; Duan, Dongsheng

    2005-01-01

    Duchenne muscular dystrophy (DMD) is the most common inherited lethal muscle degenerative disease. Currently there is no cure. Highly abbreviated microdystrophin cDNAs were developed recently for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminal-truncated ΔR4-R23/ΔC microgene (ΔR4/ΔC) has been considered as a very promising therapeutic candidate gene. In this study, we packaged a CMV.ΔR4/ΔC cassette in AAV-5 and evaluated the transduction and muscle contractile...

  13. Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element

    OpenAIRE

    Haberman, Rebecca P.; McCown, Thomas J.; Samulski, Richard Jude

    2000-01-01

    Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long...

  14. Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity

    OpenAIRE

    Haberman, Rebecca P.; Criswell, Hugh E.; Snowdy, Stephen; Ming, Zhen; Breese, George R.; Samulski, R. Jude; McCown, Thomas J.

    2002-01-01

    The N-methyl-d-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1...

  15. Reduction of experimental diabetic vascular leakage by delivery of angiostatin with a recombinant adeno-associated virus vector

    OpenAIRE

    Shyong, Mong-Ping; Lee, Fenq-Lih; Kuo, Ping-Chang; Wu, Ai-Ching; Cheng, Huey-Chung; Chen, Show-Li; Tung, Tao-Hsin; Tsao, Yeou-Ping

    2007-01-01

    Purpose To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model. Methods rAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). D...

  16. Adeno-associated Virus 9 Mediated FKRP Gene Therapy Restores Functional Glycosylation of α-dystroglycan and Improves Muscle Functions

    OpenAIRE

    Xu, Lei; Lu, Pei Juan; Wang, Chi-Hsien; Keramaris, Elizabeth; Qiao, Chunping; Xiao, Bin; Blake, Derek J.; Xiao, Xiao; Lu, Qi Long

    2013-01-01

    Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker–Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with ...

  17. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shuohao [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kawabe, Yoshinori; Ito, Akira [Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kamihira, Masamichi, E-mail: kamihira@chem-eng.kyushu-u.ac.jp [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  18. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    International Nuclear Information System (INIS)

    Highlights: ► Adeno-associated virus (AAV) is capable of targeted integration in human cells. ► Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. ► A targeted integration system of IDRV DNA using the AAV integration mechanism. ► Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  19. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    Energy Technology Data Exchange (ETDEWEB)

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Kohlbrenner, Erik [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Chiorini, John A. [GTTB, NIDCR, National Institutes of Health, Bethesda, MD 20892 (United States); McKenna, Robert [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Muzyczka, Nicholas [Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Zolotukhin, Sergei [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Agbandje-McKenna, Mavis, E-mail: mckenna@ufl.edu [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States)

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  20. Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

    Institute of Scientific and Technical Information of China (English)

    Tung-Yu Tsui; Chi-Keung Lau; Jian Ma; Gabriel Glockzin; Aiman Obed; Hans J Schlitt; Sheung-Tat Fan

    2006-01-01

    AIM: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl4-induced micronodular cirrhosis model.METHODS: Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated. 1x1012 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis.RESULTS: Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controis, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers.CONCLUSIONS: Enhancement of HO activity in the livers suppresses the development of cirrhosis.

  1. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    International Nuclear Information System (INIS)

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P212121, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress

  2. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S. (Oregon HSU)

    2012-05-24

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  3. Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal.

    Science.gov (United States)

    Wang, Lina; Yin, Zifei; Wang, Yuan; Lu, Yuan; Zhang, Daniel; Srivastava, Arun; Ling, Changquan; Aslanidi, George V; Ling, Chen

    2015-09-01

    We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3' end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA. PMID:26297494

  4. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    Science.gov (United States)

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies. PMID:26159373

  5. Induction of differentiation-associated changes in established human cells by infection with adeno-associated virus type 2.

    Science.gov (United States)

    Klein-Bauernschmitt, P; zur Hausen, H; Schlehofer, J R

    1992-01-01

    The nonpathogenic human defective parvovirus adeno-associated virus (AAV) type 2 induced differentiation-associated antigens in cells of the human leukemia cell line HL60 (CD 67), as well as in two different lines of immortalized human keratinocytes, HaCaT and HPK Ia cells (involucrin and cytokeratin 10). Simultaneously, expression of the c-myc and c-myb oncogenes and the retinoblastoma gene was down regulated whereas c-fos expression increased in infected cells. These data point to the potential of AAV to induce functions related to the differentiation pathway in different types of human cells. This phenomenon may be involved in the reported oncosuppressive properties of AAV infections. Images PMID:1318400

  6. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    DEFF Research Database (Denmark)

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari; Schratt, Gerhard

    2010-01-01

    delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming...

  7. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    NARCIS (Netherlands)

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    2008-01-01

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman prim

  8. Recombinant adeno-associated virus vector expressing angiostatin inhibits preretinal neovascularization in adult rats.

    Science.gov (United States)

    Lai, Chi-Chun; Wu, Wei-Chi; Chen, Show-Li; Sun, Ming-Hui; Xiao, Xiao; Ma, Lih; Lin, Keng-Kuo; Tsao, Yeou-Ping

    2005-01-01

    Clinically, preretinal neovascularization (PNV) induced by vessel occlusion is one of the leading causes to induce blindness. The present study was designed to determine if a recombinant adeno-associated viral vector expressing mouse angiostatin (rAAV-angiostatin) can inhibit experimental PNV in an adult Sprague-Dawley rat model. rAAV-angiostatin and rAAV-lacZ were delivered by intravitreal injections to the right and left eyes of rats. Transgenetic expression of angiostatin in the retina was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). PNV was established by rose-bengal-assisted laser-induced retinal vein occlusion 21 days after the viral injections. The total number and sizes of the neovascular tufts were analyzed 14 days after venous occlusion using retinal flat mount by fluorescein-isothiocyanate-dextran angiography. Electroretinograms (ERGs) were recorded to study any possibility of retinal toxicity of rAAV-angiostatin 3 months after the injections. Angiostatin gene expression in the retina was detectable by RT-PCR, and ERG analysis showed no reduction of b-waves in the rAAV-angiostatin-injected eyes. The number and size of neovascular tufts were significantly lower in rAAV-angiostatin-injected eyes (p = 0.001) than controls. These findings indicated that rAAV-angiostatin successfully suppressed experimental PNV, and no retinal toxicity of the rAAV-angiostatin injection was observed according to ERG recordings. PMID:15637422

  9. Efficient Replication of Adeno-Associated Virus Type 2 Vectors: a cis-Acting Element outside of the Terminal Repeats and a Minimal Size

    OpenAIRE

    Tullis, Gregory E.; Shenk, Thomas

    2000-01-01

    Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containin...

  10. A new genetic vaccine platform based on an adeno-associated virus isolated from a rhesus macaque.

    Science.gov (United States)

    Lin, Jianping; Calcedo, Roberto; Vandenberghe, Luk H; Bell, Peter; Somanathan, Suryanarayan; Wilson, James M

    2009-12-01

    We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8(+) T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8(+) T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses. PMID:19812149

  11. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    International Nuclear Information System (INIS)

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68

  12. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

    Science.gov (United States)

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research. PMID:24435020

  13. Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats

    Institute of Scientific and Technical Information of China (English)

    Sen Lu; Yue Yu; Yun Gao; Guo-Qiang Li; Xue-Hao Wang

    2008-01-01

    BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno-associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS:The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantita-tive PCR was used to measure the expression of IL-2, IFN-γ, IL-4 and IL-10 in the allografts. RESULTS:The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-γ, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62±0.09, 1.52±0.11, 1.50± 0.07 and 1.43±0.07 versus 1.29±0.09, 1.32±0.07, 1.34±0.06 and 1.35±0.04, respectively). CONCLUSIONS:pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological ifndings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.

  14. Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

    Science.gov (United States)

    Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

    2016-02-01

    Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. PMID:26284700

  15. Recombinant Adeno-Associated Virus Vector Genomes Take the Form of Long-Lived, Transcriptionally Competent Episomes in Human Muscle.

    Science.gov (United States)

    Schnepp, Bruce C; Chulay, Jeffrey D; Ye, Guo-Jie; Flotte, Terence R; Trapnell, Bruce C; Johnson, Philip R

    2016-01-01

    Gene augmentation therapy as a strategy to treat alpha-1 antitrypsin (AAT) deficiency has reached phase 2 clinical testing in humans. Sustained serum levels of AAT have been observed beyond one year after intramuscular administration of a recombinant adeno-associated virus (rAAV) vector expressing the AAT gene. In this study, sequential muscle biopsies obtained at 3 and 12 months after vector injection were examined for the presence of rAAV vector genomes. Each biopsy sample contained readily detectable vector DNA, the majority of which existed as double-stranded supercoiled and open circular episomes. Episomes persisted through 12 months, although at slightly lower levels than observed at 3 months. There was a clear dose response when comparing the low- and mid-vector-dose groups to the high-dose group. The highest absolute copy numbers were found in a high-dose subject, and serum AAT levels at 12 months confirmed that the high-dose group also had the highest sustained serum AAT levels. Sequence analysis revealed that the vast majority of episomes contained double-D inverted terminal repeats ranging from fully intact to severely deleted. Molecular clones of vector genomes derived directly from the biopsies were transcriptionally active, potentially identifying them as the source of serum AAT in the trial subjects. PMID:26650966

  16. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

    Energy Technology Data Exchange (ETDEWEB)

    DiMattia,M.; Govindasamy, L.; Levy, H.; Whitaker-Gurda, B.; Kohlbrenner, E.; Chiorini, J.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  17. Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

    International Nuclear Information System (INIS)

    Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors

  18. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

    Science.gov (United States)

    Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

    2015-10-15

    The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. PMID:26264580

  19. Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses

    Science.gov (United States)

    Bantel-Schaal, Ursula; Delius, Hajo; Schmidt, Rainer; zur Hausen, Harald

    1999-01-01

    We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy. PMID:9882294

  20. Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

    Institute of Scientific and Technical Information of China (English)

    Yonglun Luo; Emil Kofod-Olsen; Rikke Christensen; Charlotte Brandt S(φ)rensen; Lars Bolund

    2012-01-01

    Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer (SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis,hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

  1. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    Directory of Open Access Journals (Sweden)

    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

  2. Preparation of a recombinant adeno-associated virus vector encoding the human NIS gene and its expression in thyroid cancer cell lines

    International Nuclear Information System (INIS)

    Objective: To demonstrate the feasibility of thyroid gene therapy by using a adeno-associated virus vector to deliver the sodium/iodide symporter (NIS) gene into the thyroid tumor cells. Methods: The recombinant adeno-associated virus encoding the human NIS gene (rAAV-NIS, the immunofluorescence and iodide uptake studies as well as inhibited iodide uptake tests were carried out. Results: A rAAV encoding the human NIS gene was successfully prepared. Immunofluorescence analysis confirmed the expression of the NIS protein in the tumor cells, and it was localized at the cell surface and possessed a function of iodine uptake as well as suppression by perchlorates similar to the function and character with normal thyroid cell. Conclusion: rAAV-NIS is very efficient in triggering iodide uptake by infected tumor cells, and it outline the potential of this novel cancer gene therapy approach for a targted radiotherapy. (authors)

  3. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

    OpenAIRE

    Srivastava, C H; Samulski, R J; L. Lu; Larsen, S H; A Srivastava

    1989-01-01

    To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for...

  4. Cloning of adeno-associated virus into pBR322: Rescue of intact virus from the recombinant plasmid in human cells

    OpenAIRE

    1982-01-01

    We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescu...

  5. Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS

    OpenAIRE

    Lepore, Angelo C.; Haenggeli, Christine; Gasmi, Mehdi; Bishop, Kathie M.; Bartus, Raymond T.; Maragakis, Nicholas J.; Rothstein, Jeffrey D.

    2007-01-01

    The potent neuroprotective activities of neurotrophic factors, including insulin-like growth factor 1 (IGF-1), make them promising candidates for treatment of amyotrophic lateral sclerosis (ALS). In an effort to maximize rate of motor neuron transduction, achieve high levels of spinal IGF-1, and thus enhance therapeutic benefit, we injected an adeno-associated virus 2 (AAV2)-based vector encoding human IGF-1 (CERE-130) into lumbar spinal cord parenchyma of SOD1G93A mice. We observed robust an...

  6. A single injection of recombinant adeno-associated virus into the lumbar cistern delivers transgene expression throughout the whole spinal cord

    OpenAIRE

    Guo, Yansu; Wang, Dan; Qiao, Tao; Yang, Chunxing; Su, Qin; Gao, Guangping; Xu, Zuoshang

    2015-01-01

    The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in sixty to ninety percent of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells and endothelial cells. Add...

  7. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    International Nuclear Information System (INIS)

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV) vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy

  8. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    Directory of Open Access Journals (Sweden)

    Zheng Liu

    2012-04-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Methods Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. Results The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. Conclusion These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.

  9. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.

    LENUS (Irish Health Repository)

    Luo, Xiaoyan

    2011-11-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-\\/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg\\/dl) was prevented for >6 months by the dsAAV2\\/7, dsAAV2\\/8, and dsAAV2\\/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2\\/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2\\/7 and dsAAV2\\/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2\\/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-\\/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2\\/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2\\/9 vector. Hepatorenal correction in G6pase (-\\/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.

  10. Persistence, localization, and external control of transgene expression after single injection of adeno-associated virus into injured joints.

    Science.gov (United States)

    Lee, Hannah H; O'Malley, Michael J; Friel, Nicole A; Payne, Karin A; Qiao, Chunping; Xiao, Xiao; Chu, Constance R

    2013-04-01

    A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra-articular soft tissues when AAV is injected preinjury. Two AAV injection time points, postinjury and preinjury, were investigated in osteochondral defect and anterior cruciate ligament transection models of joint injury. Rats injected with AAV tetracycline response element (TRE)-luciferase received oral doxycycline for 7 days. Luciferase expression was evaluated longitudinally for 6 months. Transgene expression was persistent and controllable with oral doxycycline for 6 months in all groups. However, the location of transgene expression was different: postinjury AAV-injected knees had luciferase expression highly localized to the cartilage, while preinjury AAV-injected knees had more widespread signal from intra-articular soft tissues. The differential transgene localization between preinjury and postinjury injection can be used to optimize treatment strategies. Highly localized postinjury injection appears advantageous for treatments targeting repair cells. The more generalized and controllable reservoir of transgene expression following AAV injection before anterior cruciate ligament transection (ACLT) suggests an intriguing concept for prophylactic delivery of joint protective factors to individuals at high risk for early osteoarthritis (OA). Successful external control of intra-articular transgene expression provides an added margin of safety for these potential clinical applications. PMID:23496155

  11. In vivo evaluation of adeno-associated virus gene transfer in airways of mice with acute or chronic respiratory infection.

    Science.gov (United States)

    Myint, Melissa; Limberis, Maria P; Bell, Peter; Somanathan, Suryanarayan; Haczku, Angela; Wilson, James M; Diamond, Scott L

    2014-11-01

    Patients with cystic fibrosis (CF) often suffer chronic lung infection with concomitant inflammation, a setting that may reduce the efficacy of gene transfer. While gene therapy development for CF often involves viral-based vectors, little is known about gene transfer in the context of an infected airway. In this study, three mouse models were established to evaluate adeno-associated virus (AAV) gene transfer in such an environment. Bordetella bronchiseptica RB50 was used in a chronic, nonlethal respiratory infection in C57BL/6 mice. An inoculum of ∼10(5) CFU allowed B. bronchiseptica RB50 to persist in the upper and lower respiratory tracts for at least 21 days. In this infection model, administration of an AAV vector on day 2 resulted in 2.8-fold reduction of reporter gene expression compared with that observed in uninfected controls. Postponement of AAV administration to day 14 resulted in an even greater (eightfold) reduction of reporter gene expression, when compared with uninfected controls. In another infection model, Pseudomonas aeruginosa PAO1 was used to infect surfactant protein D (SP-D) or surfactant protein A (SP-A) knockout (KO) mice. With an inoculum of ∼10(5) CFU, infection persisted for 2 days in the nasal cavity of either mouse model. Reporter gene expression was approximately ∼2.5-fold lower compared with uninfected mice. In the SP-D KO model, postponement of AAV administration to day 9 postinfection resulted in only a two fold reduction in reporter gene expression, when compared with expression seen in uninfected controls. These results confirm that respiratory infections, both ongoing and recently resolved, decrease the efficacy of AAV-mediated gene transfer. PMID:25144316

  12. Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

    Directory of Open Access Journals (Sweden)

    Jennifer L Daily

    Full Text Available Angelman syndrome (AS, a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286 and Thr(305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

  13. Liver-Specific Allergen Gene Transfer by Adeno-Associated Virus Suppresses Allergic Airway Inflammation in Mice.

    Science.gov (United States)

    Chan, Cheng-Chi; Lai, Chin-Wen; Wu, Chia-Jen; Chen, Li-Chen; Tao, Mi-Hua; Kuo, Ming-Ling

    2016-08-01

    Allergic airway inflammation driven by T helper 2 (Th2)-type immunity is characterized by airway hyperresponsiveness, eosinophilic infiltration, and elevated IgE production. Various novel strategies for managing asthma have been explored, such as DNA vaccines, T-cell peptides, and allergen-specific immunotherapy. A principal goal of most immunotherapeutic approaches is active and long-term allergen-specific tolerance. Liver-specific gene transfer using adeno-associated virus (AAV) has been shown to favorably induce tolerogenic responses to therapeutic products in various experimental models. AAV8 has strong liver tropism and induces immune tolerance in mice. The present study aimed to determine whether hepatocyte-specific allergen expression by pseudotyped AAV2/8 alleviates asthmatic symptoms in ovalbumin (OVA)-sensitized mice. Mice were intravenously injected with AAV2/8 vector carrying membrane-bound OVA transgene under transcriptional control of a hepatocyte-specific alpha 1 antitrypsin promoter (AAV2/8-OVA) and then sensitized with OVA. AAV2/8-OVA specifically transduced the OVA transgene in the liver. Airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and Th2 cytokines were significantly suppressed in both the lungs and secondary lymphoid organs of asthmatic mice infected with AAV2/8-OVA. Significant reduction of OVA-specific antibodies was detected in the bronchoalveolar lavage fluid from AAV2/8-OVA-treated mice. Moreover, AAV2/8-OVA treatment prominently promoted the expression of Foxp3, IL-10, and TGF-β in the liver. Enhanced Foxp3 expression was also detected in the lungs of asthmatic mice after AAV2/8-OVA treatment. Taken together, these results suggest that the induction of immune tolerance by hepatic AAV gene transfer may be beneficial for modulating allergic asthma. PMID:27178525

  14. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity

    International Nuclear Information System (INIS)

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process

  15. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    International Nuclear Information System (INIS)

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

  16. Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-he; KE Xiao-mei; QIN Yong; GU Zhi-ping; XIAO Shui-fang

    2007-01-01

    Background Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-xL is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-xL as a therapeutic agent in the murine model of aminoglycoside ototoxicity.Methods Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-xL gene was injected into mice cochleae prior to injection of kanamycin. Bcl-xL expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function.Results The animals in the AAV2-Bcl-xL/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side.Conclusions AAV2-Bcl-xL afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-xL might be an approach with respect to potential therapeutic application in the cochlear degeneration.

  17. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Directory of Open Access Journals (Sweden)

    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  18. The X gene of adeno-associated virus 2 (AAV2 is involved in viral DNA replication.

    Directory of Open Access Journals (Sweden)

    Maohua Cao

    Full Text Available Adeno-associated virus (AAV (type 2 is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393, overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81. Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91, a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5 or Ad5 helper plasmid (pHelper. The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects. Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold. Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  19. Activation of the NF-κB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy

    OpenAIRE

    Jayandharan, Giridhara R.; Aslanidi, George; Martino, Ashley T.; Jahn, Stephan C.; Perrin, George Q.; Herzog, Roland W.; Srivastava, Arun

    2011-01-01

    Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold...

  20. A Novel Gene Expression Control System and Its Use in Stable, High-Titer 293 Cell-Based Adeno-Associated Virus Packaging Cell Lines

    OpenAIRE

    Qiao, Chunping; Bing WANG; Zhu, Xiaodong; Li, Juan; Xiao, Xiao

    2002-01-01

    Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene ex...

  1. Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy

    OpenAIRE

    Xiao, Xiao; Li, Juan; Tsao, Yeou-Ping; Dressman, Devin; Hoffman, Eric P; Watchko, Jon F.

    2000-01-01

    Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose A...

  2. Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture

    Directory of Open Access Journals (Sweden)

    Kitto Kelley F

    2010-05-01

    Full Text Available Abstract Background Neuronal transduction by adeno-associated viral (AAV vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG and spinal cord offers substantial opportunities to 1 further study mechanisms underlying chronic pain, and 2 develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5- and AAV serotype 8 (AAV8-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Results Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP or AAV8 (rAAV8-GFP carrying the green fluorescent protein (GFP gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1 GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2 dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4 -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP. Conclusions The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.

  3. Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

    Science.gov (United States)

    Silver, Jared N; Elder, Melissa; Conlon, Thomas; Cruz, Pedro; Wright, Amy J; Srivastava, Arun; Flotte, Terence R

    2011-08-01

    Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010

  4. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.

    Science.gov (United States)

    Jin, Lei; Lange, Wienke; Kempmann, Annika; Maybeck, Vanessa; Günther, Anne; Gruteser, Nadine; Baumann, Arnd; Offenhäusser, Andreas

    2016-09-10

    In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons. PMID:27416794

  5. Silencing of T lymphocytes by antigen-driven programmed death in recombinant adeno-associated virus vector–mediated gene therapy

    Science.gov (United States)

    Velazquez, Victoria M.; Bowen, David G.

    2009-01-01

    Recombinant adeno-associated virus (rAAV) vectors are considered promising for human gene replacement because they facilitate stable expression of therapeutic proteins in transduced tissues. Whether the success of gene therapy will be influenced by cellular immune responses targeting transgene-encoded proteins that are potentially immunogenic is unknown. Here we characterized CD8+ T-cell activity against β-galactosidase and enhanced green fluorescent protein, model antigens containing major histocompatibility complex (MHC) class I epitopes that are constitutively produced in murine skeletal muscle after rAAV vector transduction. Antigen-specific CD8+ T cells were detected in the spleen and liver of mice within 7 days of muscle transduction. CD8+ T-cell frequencies in these organs were stable, and effector functions were intact for months despite ongoing antigen production in muscle. CD8+ T cells also infiltrated transduced muscle, where frequencies were at least 5-fold higher than in untransduced spleen and liver. Significantly, the majority of antigen-specific CD8+ T cells in vector-transduced muscle were not functional. Loss of function in the muscle was associated with programmed death of the effector cells. Stable gene expression therefore depended on selective death of CD8+ T cells at the site of antigen production, an effective mechanism for subverting immunity that is also potentially reversible. PMID:18566327

  6. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Directory of Open Access Journals (Sweden)

    Lina Li

    Full Text Available Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA. CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9 Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  7. Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

    Science.gov (United States)

    Tomono, Taro; Hirai, Yukihiko; Okada, Hironori; Adachi, Kumi; Ishii, Akiko; Shimada, Takashi; Onodera, Masafumi; Tamaoka, Akira; Okada, Takashi

    2016-01-01

    Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy. PMID:26913289

  8. Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

    Science.gov (United States)

    Heller, Kristin N; Montgomery, Chrystal L; Shontz, Kimberly M; Clark, K Reed; Mendell, Jerry R; Rodino-Klapac, Louise R

    2015-10-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD. PMID:26076707

  9. Adeno-associated virus type 2 rep protein inhibits human papillomavirus type 16 E2 recruitment of the transcriptional coactivator p300.

    Science.gov (United States)

    Marcello, A; Massimi, P; Banks, L; Giacca, M

    2000-10-01

    Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300. PMID:10982355

  10. HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

    International Nuclear Information System (INIS)

    Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the β-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms

  11. Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor.

    Science.gov (United States)

    Ponnazhagan, Selvarangan; Mahendra, Gandham; Kumar, Sanjay; Shaw, Denise R; Stockard, Cecil R; Grizzle, William E; Meleth, Sreelatha

    2004-03-01

    Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy. PMID:14996740

  12. Pre-existing immunity to adeno-associated virus (AAV)2 limits transgene expression following intracerebral AAV2-based gene delivery in a 6-hydroxydopamine model of Parkinson's disease.

    OpenAIRE

    Janelidze, Shorena; Nordström, Ulrika; Kügler, Sebastian; Brundin, Patrik

    2014-01-01

    BACKGROUND: Adeno-associated virus (AAV) vectors are used to deliver potentially therapeutic genes in clinical trials in Parkinson's disease (PD). Pre-existing immunity to AAV and a local neuroinflammatory response might negatively affect the efficacy of such AAV-mediated gene delivery. METHODS: We pre-immunized rats with wild-type AAV-2. Three months later, we created PD-like lesions by intrastriatal injections of 6-hydroxydopamine (6-OHDA) in 50% of the animals. One month later, we inj...

  13. Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

    Science.gov (United States)

    Gomez, Eric J; Gerhardt, Karl; Judd, Justin; Tabor, Jeffrey J; Suh, Junghae

    2016-01-26

    Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts. PMID:26618393

  14. Development of Recombinant Adeno-Associated Virus Serotype 2/8 Carrying Kringle Domains of Human Plasminogen for Sustained Expression and Cancer Therapy.

    Science.gov (United States)

    Kuo, Cheng-Hsiang; Chang, Bi-Ing; Lee, Fang-Tzu; Chen, Po-Ku; Lee, Jeng-Shin; Shi, Guey-Yueh; Wu, Hua-Lin

    2015-09-01

    Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1-5 (K1-5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1-5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1-5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1-5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development. PMID:25950911

  15. A Novel Gene Expression Control System and Its Use in Stable, High-Titer 293 Cell-Based Adeno-Associated Virus Packaging Cell Lines

    Science.gov (United States)

    Qiao, Chunping; Wang, Bing; Zhu, Xiaodong; Li, Juan; Xiao, Xiao

    2002-01-01

    Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene expression since its promoter is located within the coding sequence of Rep78/68. To tightly regulate all four Rep proteins by using their own promoters, we have developed a novel gene control paradigm termed “dual splicing switch,” which disrupts all four Rep genes by inserting into their shared coding region an intron that harbors transcription termination sequences flanked the LoxP sites. As a result, the structure and activities of the Rep gene promoters, both p5 and p19, are not affected; however, all of the Rep transcripts are prematurely terminated and the genes were inactivated. Removal of the terminator by Cre protein reactivates the transcription of all four Rep proteins derived from their own promoters. This switch system was initially tested in the lacZ gene and a 600-fold induction of β-galactosidase activity was observed. Using the dual splicing switch strategy, we have subsequently established a number of AAV packaging cell lines from 293 cells, which showed a normal growth rate, high stability, and more importantly, high yields of AAV vectors. Such a gene control paradigm is also useful for other viruses, e.g., autonomous parvoviruses. Finally, the high-titer 293-based AAV packaging cell lines should greatly reduce the risk of wild-type adenovirus contamination and provide a scalable AAV vector production method for both preclinical and clinical studies. PMID:12438627

  16. Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Amin Hajitou

    2013-10-01

    Full Text Available Bacteriophage (phage, viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV. This novel AAV/phage hybrid (AAVP specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  17. Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene.

    Science.gov (United States)

    Mueller, Christian; Torrez, Daniel; Braag, Sofia; Martino, Ashley; Clarke, Tracy; Campbell-Thompson, Martha; Flotte, Terence R

    2008-01-01

    Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes. PMID:18023072

  18. An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

    Directory of Open Access Journals (Sweden)

    Abdelwahed Chtarto

    Full Text Available Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS. This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

  19. Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells

    International Nuclear Information System (INIS)

    Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection

  20. Intracerebral adeno-associated virus gene delivery of apolipoprotein E2 markedly reduces brain amyloid pathology in Alzheimer's disease mouse models.

    Science.gov (United States)

    Zhao, Lingzhi; Gottesdiener, Andrew J; Parmar, Mayur; Li, Mingjie; Kaminsky, Stephen M; Chiuchiolo, Maria J; Sondhi, Dolan; Sullivan, Patrick M; Holtzman, David M; Crystal, Ronald G; Paul, Steven M

    2016-08-01

    The common apolipoprotein E alleles (ε4, ε3, and ε2) are important genetic risk factors for late-onset Alzheimer's disease, with the ε4 allele increasing risk and reducing the age of onset and the ε2 allele decreasing risk and markedly delaying the age of onset. Preclinical and clinical studies have shown that apolipoprotein E (APOE) genotype also predicts the timing and amount of brain amyloid-β (Aβ) peptide deposition and amyloid burden (ε4 >ε3 >ε2). Using several administration protocols, we now report that direct intracerebral adeno-associated virus (AAV)-mediated delivery of APOE2 markedly reduces brain soluble (including oligomeric) and insoluble Aβ levels as well as amyloid burden in 2 mouse models of brain amyloidosis whose pathology is dependent on either the expression of murine Apoe or more importantly on human APOE4. The efficacy of APOE2 to reduce brain Aβ burden in either model, however, was highly dependent on brain APOE2 levels and the amount of pre-existing Aβ and amyloid deposition. We further demonstrate that a widespread reduction of brain Aβ burden can be achieved through a single injection of vector via intrathalamic delivery of AAV expressing APOE2 gene. Our results demonstrate that AAV gene delivery of APOE2 using an AAV vector rescues the detrimental effects of APOE4 on brain amyloid pathology and may represent a viable therapeutic approach for treating or preventing Alzheimer's disease especially if sufficient brain APOE2 levels can be achieved early in the course of the disease. PMID:27318144

  1. A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

    Science.gov (United States)

    Gomes, Michelle M; Wang, Lingyan; Jiang, Han; Kahl, Christoph A; Brigande, John V

    2016-01-01

    There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders. PMID:27259920

  2. Infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins.

    Science.gov (United States)

    Hermanns, J; Schulze, A; Jansen-Db1urr, P; Kleinschmidt, J A; Schmidt, R; zur Hausen, H

    1997-01-01

    It has been demonstrated that infection of primary human cells with adeno-associated viruses (AAV) leads to a decrease in cellular proliferation and to growth arrest. We analyzed the molecular basis of this phenomenon and observed that infection with AAV type 2 (AAV2) had an effect on several factors engaged in the control of the mammalian cell cycle. In particular, all of the pRB family members, pRB, p107, and p130, which are involved in G1 cell cycle checkpoint control, were affected. After infection, a shift from hyper- to hypophosphorylated forms was observed. Cyclins A and B1, which are required for G1/S transition and progression into mitosis, respectively, were downregulated at the transcriptional level as well as at the protein level, whereas the G1 cyclins D1 and E remained unaffected. In addition, the steady-state levels of cyclin-dependent kinases CDK1 and CDK2 and of transcription factor E2F-1 were diminished. Of all the factors known to be involved in phosphorylation of pRB family proteins, only the CDK inhibitor p21WAF1 exhibited a response to AAV2 infection. p21WAF1 mRNA was quickly and progressively upregulated in a p53-independent manner over at least 72 h. Consistent with the increased p21WAF1 protein levels, cyclin E- and cyclin A-dependent kinase activities declined to low levels and E2F-p130-cyclin-CDK2 complexes were disrupted. From these data, we conclude that the major effect of AAV2 infection on primary human fibroblasts appears to be upregulation of p21WAF1 gene expression and thus cell cycle arrest by the suppression of pRB family protein phosphorylation. PMID:9223493

  3. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  4. Adeno-Associated Virus Overexpression of Angiotensin-Converting Enzyme-2 Reverses Diabetic Retinopathy in Type 1 Diabetes in Mice.

    Science.gov (United States)

    Dominguez, James M; Hu, Ping; Caballero, Sergio; Moldovan, Leni; Verma, Amrisha; Oudit, Gavin Y; Li, Qiuhong; Grant, Maria B

    2016-06-01

    Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system. PMID:27178803

  5. Avian Influenza A Virus Infections in Humans

    Science.gov (United States)

    ... Past Newsletters Avian Influenza A Virus Infections in Humans Language: English Español Recommend on Facebook Tweet ... A Viruses Avian Influenza A Virus Infections in Humans Although avian influenza A viruses usually do not ...

  6. Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Tandon Apurva

    2011-08-01

    Full Text Available Abstract Background In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2 induces apoptosis in Human Papillomavirus (HPV positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive, as well as MDA-MB-231 (highly invasive human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs isolated from tissue biopsies of patients undergoing breast reduction surgery. Results AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. Conclusion AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated

  7. Expression of human nerve growth factor β gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector

    Institute of Scientific and Technical Information of China (English)

    高凯; 吴勇杰; 吴小兵; 饶春明; 王军志

    2004-01-01

    Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV-2 vector (rAAV-2/hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFβ. rAAV-2/hNGFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.Results After 48 hours, hNGFβ content in supernatant was up to (188.0±28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFβ at multiplicity of infection (MOI)1.0×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFβ. Whole brain hNGFβ content in rAAV-2/hNGFβ transferred group was up to (636.2±140.6) pg/ml. hNGFβ content of BBB disruption in rAAV-2/hNGFβ infused group increased significantly compared to the control group (P<0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.Conclusion rAAV-2/hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

  8. Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.

    Directory of Open Access Journals (Sweden)

    Barbara Bogner

    Full Text Available Adeno associated virus (AAV is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV's ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc genomes in the anterior segment of the eye.AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE, iris and chamber angle including trabecular meshwork, with scAAV2(Y444F and scAAV2(triple being the most efficient.This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene

  9. Avian influenza viruses in humans.

    OpenAIRE

    Malik Peiris, J S

    2009-01-01

    Past pandemics arose from low pathogenic avian influenza (LPAI) viruses. In more recent times, highly pathogenic avian influenza (HPAI) H5N1, LPAI H9N2 and both HPAI and LPAI H7 viruses have repeatedly caused zoonotic disease in humans. Such infections did not lead to sustained human-to-human transmission. Experimental infection of human volunteers and seroepidemiological studies suggest that avian influenza viruses of other subtypes may also infect humans. Viruses of the H7 subtype appear to...

  10. A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

    Directory of Open Access Journals (Sweden)

    Fengqin Zhu

    Full Text Available More than 170 million individuals worldwide are infected with hepatitis C virus (HCV, and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

  11. Avian influenza virus RNA extraction

    Science.gov (United States)

    The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

  12. Recombinant adeno-associated virus-mediated human kallikrein gene therapy prevents high-salt diet-induced hypertension without effect on basal blood pressure

    Institute of Scientific and Technical Information of China (English)

    Jiang-tao YAN; Tao WANG; Juan LI; Xiao XIAO; Dao-wen WANG

    2008-01-01

    Aim: To investigate the effects of the expression of human kallikrein (HK) on basal level blood pressure and high-salt diet-induced hypertension. Methods: We delivered the recombinant adeno-associated viral (rAAV)-mediated HK (rAAV-HK) gene and rAAV-LacZ (as the control) to normal, adult Sprague-Dawley rats. The animals were administered a normal diet in the first 4 weeks, followed by a high-salt diet. The expression of HK in the rats was assessed by ELISA and RT-PCR. Blood pressure and Na~ and K~ urinary excretion were monitored. Results: Under the normal diet, no obvious changes in blood pressure and Na+ and K+ urinary excretion were observed. When the high-salt diet was administered, sys-tolic blood pressure in the control animals receiving rAAV-LacZ increased from 122.3±1. 13 mmHg to a stable 142.4±1.77 mmHg 8 weeks after the high-salt diet. In contrast, there was no significant increase in the blood pressure in the rAAV-HK-treated group, in which the blood pressure remained at 121.9±1.73 mmHg. In the rAAV-HK-treated group, Na+ and K+ urinary excretion were higher compared to those of the control group. The morphological analysis showed that HK delivery remarkably protected against renal damage induced by a high-salt intake. Conclusion: Our study indicates that rAAV-mediated human tissue kallikrein gene delivery is a potentially safe method for the long-term treatment of hypertension. More importantly, it could be applied in the salt-sensitive population to prevent the occurrence of hypertension.

  13. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.

    Science.gov (United States)

    Karumuthil-Melethil, Subha; Nagabhushan Kalburgi, Sahana; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D; Keimel, John G; Mark, Brian L; Mahuran, Don; Walia, Jagdeep S; Gray, Steven J

    2016-07-01

    GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α-β), "A" isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP- GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter-intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the nervous system

  14. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers.

    Science.gov (United States)

    Yan, Ziying; Sun, Xingshen; Feng, Zehua; Li, Guiying; Fisher, John T; Stewart, Zoe A; Engelhardt, John F

    2015-06-01

    The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRΔR). This vector yielded an ∼17-fold increase in expression of CFTR and significantly improved Cl(-) currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway. PMID:25763813

  15. [Establishment of hepatitis B virus (HBV) chronic infection mouse model by in vivo transduction with a recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome (rAAN8-1. 3HBV)].

    Science.gov (United States)

    Dong, Xiao-Yan; Yu, Chi-Jie; Wang, Gang; Tian, Wen-Hong; Lu, Yue; Zhang, Feng-Wei; Wang, Wen; Wang, Yue; Tan, Wen-Jie; Wu, Xiao-Bing

    2010-11-01

    In this report, we developed a HBV infection model in C57BL/6 mouse line by in vivo injection of a recombinant adeno-associated virus 8 vector carrying 1. 3 copies of HBV genome (ayw subtype) (rAAV8-1. 3HBV). We firstly prepared and purified the rAAV8-1. 3HBV and then injected it into three C57BL/6 mice with the dose of 2 x 10e11vg, respectively. HBsAg and HBeAg were assayed in sera collected at different time points post injection. Ten weeks post injection, the three mice were sacrificed and blood and liver tissue were taken for assay. Copies of HBV DNA were detected by real time PCR and the way of HBV DNA replication was identified by PCR. Subsequently, detection of HBV antigen by immunohistochemistry and pathology analysis of liver tissue of mice were performed. The results suggested that expression of HBsAg and HBeAg lasted for at least 10 weeks in mice sera. Among mice injected with rAAV8-1. 3HBV, HBsAg levels were showed an 'increasing-decreasing-increasing' pattern (the lowest level at the 4th week post injection), while HBeAg levels were kept high and relatively stable. HBV DNA copies were 4.2 x 10(3), 3.6 x 10(3), 2.5 x 10(3) copies/mL in sera and 8.0 x 10(6), 5.7 x 10(6), 2.6 x 10(6) copies/g in hepatic tissues of three mice, respectively. We found that the linear 1. 3HBV DNA in the rAAV8-1. 3HBV could self form into circular HBV genome and replicate in livers of HBV transfected mice. HBsAg and HBcAg were both positive in liver tissue of mice injected with rAAV8-1. 3HBV and no obvious pathological characters were found in liver of mice injected with rAAV8-1. 3HBV. In conclusion, we successfully developed a HBV chronic infection model in C57BL/6 mouse line by in vivo transduction with the recombinant virus rAAV8-1. 3HBV, in which HBV genes could be continuously expressed and replicated over 10 weeks, and paved a way for further characterization of the human chronic hepatitis B virus infection and evaluation of vaccine and anti-HBV agents. PMID:21344744

  16. Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

    OpenAIRE

    Copeland, N G; Cooper, G M

    1980-01-01

    Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.

  17. Molecular characterization of Indonesia avian influenza virus

    Directory of Open Access Journals (Sweden)

    N.L.P.I. Dharmayanti

    2005-06-01

    Full Text Available Avian influenza outbreaks in poultry have been reported in Java island since August 2003. A total of 14 isolates of avian influenza virus has been isolated from October 2003 to October 2004. The viruses have been identified as HPAI H5N1 subtype. All of them were characterized further at genetic level and also for their pathogenicity. Phylogenetic analysis showed all of the avian influenza virus isolates were closely related to avian influenza virus from China (A/Duck/China/E319-2/03(H5N1. Molecular basis of pathogenicity in HA cleavage site indicated that the isolates of avian influenza virus have multiple basic amino acid (B-X-B-R indicating that all of the isolates representing virulent avian influenza virus (highly pathogenic avian influenza virus.

  18. Intracranial delivery of interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway.

    Science.gov (United States)

    Yang, Junling; Kou, Jinghong; Lim, Jeong-Eun; Lalonde, Robert; Fukuchi, Ken-Ichiro

    2016-03-01

    Interleukin-17A (IL-17A) is generally considered as one of the pathogenic factors involved in multiple sclerosis (MS). Indirect evidence for this is that IL-17A-producing T helper 17 (Th17) cells preferentially accumulate in lesions of MS and experimental autoimmune encephalomyelitis (EAE). However, a direct involvement of IL-17A in MS pathogenesis is still an open question. In this study, we overexpressed IL-17A in the brains of mice (IL-17A-in-Brain mice) via recombinant adeno-associated virus serotype 5 (rAAV5)-mediated gene delivery. In spite of high levels of IL-17A expression in the brain and blood, IL-17A-in-Brain mice exhibit no inflammatory responses and no abnormalities in motor coordination and spatial orientation. Unexpectedly, IL-17A-in-Brain mice show decreases in body weight and adipose tissue mass and an improvement in glucose tolerance and insulin sensitivity. IL-17A enhances glucose uptake in PC12 cells by activation of AKT. Our results provide direct evidence for the first time that IL-17A overexpression in the central nervous system does not cause physical and learning disabilities and neuroinflammation and suggest that IL-17A may regulate glucose metabolism through the AKT signaling pathway. PMID:26562537

  19. Avian influenza virus in pregnancy.

    Science.gov (United States)

    Liu, Shelan; Sha, Jianping; Yu, Zhao; Hu, Yan; Chan, Ta-Chien; Wang, Xiaoxiao; Pan, Hao; Cheng, Wei; Mao, Shenghua; Zhang, Run Ju; Chen, Enfu

    2016-07-01

    The unprecedented epizootic of avian influenza viruses, such as H5N1, H5N6, H7N1 and H10N8, has continued to cause disease in humans in recent years. In 2013, another novel influenza A (H7N9) virus emerged in China, and 30% of those patients died. Pregnant women are particularly susceptible to avian influenza and are more likely to develop severe complications and to die, especially when infection occurs in the middle and late trimesters. Viremia is believed to occur infrequently, and thus vertical transmission induced by avian influenza appears to be rare. However, avian influenza increases the risk of adverse pregnancy outcomes, including spontaneous abortion, preterm birth and fatal distress. This review summarises 39 cases of pregnant women and their fetuses from different countries dating back to 1997, including 11, 15 and 13 infections with H7N9, H5N1 and the 2009 pandemic influenza (H1N1), respectively. We analysed the epidemic features, following the geographical, population and pregnancy trimester distributions; underlying diseases; exposure history; medical timelines; human-to-human transmission; pathogenicity and vertical transmission; antivirus treatments; maternal severity and mortality and pregnancy outcome. The common experiences reported in different countries and areas suggest that early identification and treatment are imperative. In the future, vigilant virologic and epidemiologic surveillance systems should be developed to monitor avian influenza viruses during pregnancy. Furthermore, extensive study on the immune mechanisms should be conducted, as this will guide safe, rational immunomodulatory treatment among this high-risk population. Most importantly, we should develop a universal avian influenza virus vaccine to prevent outbreaks of the different subtypes. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27187752

  20. Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

    OpenAIRE

    Wang, X S; Yoder, M C; Zhou, S. Z.; A Srivastava

    1995-01-01

    The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B1...

  1. Comparison of Efficacy of the Disease-Specific LOX1- and Constitutive Cytomegalovirus-Promoters in Expressing Interleukin 10 through Adeno-Associated Virus 2/8 Delivery in Atherosclerotic Mice

    Science.gov (United States)

    Zhu, Hongqing; Cao, Maohua; Mirandola, Leonardo; Figueroa, Jose A.; Cobos, Everardo; Chiriva-Internati, Maurizio; Hermonat, Paul L.

    2014-01-01

    The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of “disease-specific promoters” has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2) using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery. PMID:24736312

  2. Comparison of efficacy of the disease-specific LOX1- and constitutive cytomegalovirus-promoters in expressing interleukin 10 through adeno-associated virus 2/8 delivery in atherosclerotic mice.

    Directory of Open Access Journals (Sweden)

    Hongqing Zhu

    Full Text Available The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of "disease-specific promoters" has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2 using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery.

  3. In vitro evaluation of mitochondrial dysfunction and treatment with adeno-associated virus vector in fibroblasts from Doberman Pinschers with dilated cardiomyopathy and a pyruvate dehydrogenase kinase 4 mutation.

    Science.gov (United States)

    Sosa, Ivan; Estrada, Amara H; Winter, Brandy D; Erger, Kirsten E; Conlon, Thomas J

    2016-02-01

    OBJECTIVE To compare mitochondrial oxygen consumption rate (OCR) of fibroblasts from Doberman Pinschers with and without dilated cardiomyopathy (DCM) and mutation of the gene for pyruvate dehydrogenase kinase isozyme 4 (PDK4) and to evaluate in vitro whether treatment with adeno-associated virus (AAV) vector (ie, gene therapy) would alter metabolic efficiency. ANIMALS 10 Doberman Pinschers screened for DCM and PDK4 mutation. PROCEDURES Fibroblasts were harvested from skin biopsy specimens obtained from Doberman Pinschers, and dogs were classified as without DCM or PDK4 mutation (n = 3) or with occult DCM and heterozygous (4) or homozygous (3) for PDK4 mutation. Fibroblasts were or were not treated with tyrosine mutant AAV type 2 vector containing PDK4 at multiplicities of infection of 1,000. Mitochondrial OCR was measured to evaluate mitochondrial metabolism. The OCR was compared among dog groups and between untreated and treated fibroblasts within groups. RESULTS Mean ± SD basal OCR of fibroblasts from heterozygous (74 ± 8 pmol of O2/min) and homozygous (58 ± 12 pmol of O2/min) dogs was significantly lower than that for dogs without PDK4 mutation (115 ± 9 pmol of O2/min). After AAV transduction, OCR did not increase significantly in any group (mutation-free group, 121 ± 26 pmol of O2/min; heterozygous group, 88 ± 6 pmol of O2/min; homozygous group, 59 ± 3 pmol of O2/min). CONCLUSIONS AND CLINICAL RELEVANCE Mitochondrial function was altered in skin fibroblasts of Doberman Pinschers with DCM and PDK4 mutation. Change in mitochondrial function after in vitro gene therapy at the multiplicities of infection used in this study was not significant. (Am J Vet Res 2016;77:156-161). PMID:27027709

  4. Molecular characterization of Indonesia avian influenza virus

    OpenAIRE

    N.L.P.I Dharmayanti; R Damayanti; R Indriani; A Wiyono; R.M.A Adjid

    2005-01-01

    Avian influenza outbreaks in poultry have been reported in Java island since August 2003. A total of 14 isolates of avian influenza virus has been isolated from October 2003 to October 2004. The viruses have been identified as HPAI H5N1 subtype. All of them were characterized further at genetic level and also for their pathogenicity. Phylogenetic analysis showed all of the avian influenza virus isolates were closely related to avian influenza virus from China (A/Duck/China/E319-2/03(H5N1). Mo...

  5. Molecular patterns of avian influenza A viruses

    Institute of Scientific and Technical Information of China (English)

    KOU Zheng; LEI FuMin; WANG ShengYue; ZHOU YanHong; LI TianXian

    2008-01-01

    Avian influenza A viruses could get across the species barrier and be fatal to humans. Highly patho-genic avian influenza H5N1 virus was an example. The mechanism of interspecies transmission is not clear as yet. In this research, the protein sequences of 237 influenza A viruses with different subtypes were transformed into pseudo-signals. The energy features were extracted by the method of wavelet packet decomposition and used for virus classification by the method of hierarchical clustering. The clustering results showed that five patterns existed in avian influenza A viruses, which associated with the phenotype of interspecies transmission, and that avian viruses with patterns C and E could across species barrier and those with patterns A, B and D might not have the abilities. The results could be used to construct an early warning system to predict the transmissibility of avian influenza A viruses to humans.

  6. 重组腺相关病毒2型/人凝血因子IX的质量研究%Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    高凯; 王军志; 饶春明; 吴小兵

    2003-01-01

    目的研究并建立重组腺相关病毒2型/人凝血因子IX(recombinant adeno-associated virus type 2/human blood coagulation factor IX,rAAV-2/hFIX)的质量标准.方法采用PCR法确认病毒所携带的重组核酸结构和测定辅助病毒(helper virus)和野生型腺相关病毒(wtAAV)的残留片段.SDS-PAGE电泳测定病毒外壳蛋白分子量及纯度,TSK gel SP-NPR阳离子交换柱系统测定病毒颗粒纯度.以斑点杂交法测定病毒颗粒数.一期法于IX因子基因剔除小鼠体内测定rAAV-2/hFIX生物学活性,并通过ELISA法测定感染BHK-21细胞后hFIX的表达量.结果 PCR法确证病毒的重组核酸结构与构建预期相同;在1×107 VG的rAAV-2/hFIX颗粒中,残留辅助病毒的基因片段数少于1个拷贝;在1×108 VG的rAAV-2/hFIX颗粒中,野生型AAV-2基因片段数少于1个拷贝.病毒颗粒及外壳蛋白纯度均大于98%,病毒颗粒数大于1.0×1015 VG*L-1(virus genome*L-1).IX因子剔除小鼠肌肉注射病毒后21 d,小鼠血液中人凝血因子IX活性达到大于正常人因子IX活性的15%,IX因子的体外表达水平大于20.0 μg*L-1.其他各项检测指标均符合规定.结论建立了rAAV-2/hFIX的质量标准,用于控制产品质量.

  7. Adeno-associated viral vector transduction of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stender, Stefan; Murphy, Mary; O'Brien, Tim;

    2007-01-01

    Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector for...

  8. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector.

    Science.gov (United States)

    Grieger, Joshua C; Soltys, Stephen M; Samulski, Richard Jude

    2016-02-01

    Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal

  9. Avian Influenza Virus: The Threat of A Pandemic

    OpenAIRE

    Shih-Cheng Chang; Yi-Ying Cheng; Shin-Ru Shih

    2006-01-01

    The 1918 influenza A virus pandemic caused a death toll of 40~50 million. Currently,because of the widespread dissemination of the avian influenza virus (H5N1), there is a highrisk of another pandemic. Avian species are the natural hosts for numerous subtypes ofinfluenza A viruses; however, the highly pathogenic avian influenza virus (HPAI) is not onlyextremely lethal to domestic avian species but also can infect humans and cause death. Thisreview discusses why the avian influenza virus is co...

  10. A brief introduction to avian influenza virus

    Science.gov (United States)

    Avian influenza virus (AIV) causes a disease of high economic importance for poultry production worldwide. The earliest recorded cases of probable high pathogenicity AIV in poultry were reported in Italy in the 1870’s and avian influenza been recognized in domestic poultry through the modern era of ...

  11. Cell killing by avian leukosis viruses.

    OpenAIRE

    Weller, S K; Temin, H M

    1981-01-01

    Infection of chicken cells with a cytopathic avian leukosis virus resulted in the detachment of killed cells from the culture dish. The detached, dead cells contained more unintegrated viral DNA than the attached cells. These results confirm the hypothesis that cell killing after infection with a cytopathic avian leukosis virus is associated with accumulation of large amounts of unintegrated viral DNA. No accumulation of large amounts of integrated viral DNA was found in cells infected with c...

  12. Immunology of avian influenza virus: a review.

    Science.gov (United States)

    Suarez, D L; Schultz-Cherry, S

    2000-01-01

    Avian influenza virus can cause serious disease in a wide variety of birds and mammals, but its natural host range is in wild ducks, gulls, and shorebirds. Infections in poultry can be inapparent or cause respiratory disease, decreases in production, or a rapidly fatal systemic disease known as highly pathogenic avian influenza (HPAI). For the protection of poultry, neutralizing antibody to the hemagglutinin and neuraminidase proteins provide the primary protection against disease. A variety of vaccines elicit neutralizing antibody, including killed whole virus vaccines and fowl-pox recombinant vaccines. Antigenic drift of influenza viruses appears to be less important in causing vaccine failures in poultry as compared to humans. The cytotoxic T lymphocyte response can reduce viral shedding in mildly pathogenic avian influenza viruses, but provides questionable protection against HPAI. Influenza viruses can directly affect the immune response of infected birds, and the role of the Mx gene, interferons, and other cytokines in protection from disease remains unknown. PMID:10717293

  13. Avian Influenza A (H7N9) Virus

    Science.gov (United States)

    ... Research Making a Candidate Vaccine Virus Related Links Influenza Types Seasonal Avian Swine Variant Pandemic Other Get ... Submit What's this? Submit Button Past Newsletters Avian Influenza A (H7N9) Virus Language: English Español Recommend ...

  14. 重组8型腺相关病毒介导HBV急性感染树鼩模型建立%Establishment of a tree shrew model of acute hepatitis B virus infection by transduction with a recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome

    Institute of Scientific and Technical Information of China (English)

    曾扬; 吴小红; 胡靓雅; 刘晨风; 于虹; 郭彦; 周勇; 孙世惠; 周育森

    2013-01-01

    目的 利用重组8型腺相关病毒介导1.3拷贝HBV基因组(1.3HBV,ayw亚型)在树鼩肝脏表达,建立HBV急性感染树鼩模型.方法 通过大腿内侧静脉注射将携带有1.3 HBV的重组8型腺相关病毒(recombinant adeno-associated virus 8,rAAV8-1.3HBV)导入树鼩肝脏,通过ELISA检测树鼩血清中HBsAg、HBeAg、HBsAb、HBeAb、HBcAb,荧光定量PCR检测树鼩肝脏和血清中HBV DNA,全自动生化分析仪检测血清中ALT水平,并观察感染后肝脏的病变情况.结果 HBV感染主要血清标志物1~2周内均检测阳性;30 d后肝组织仍可检测到病毒抗原阳性细胞;55 d时肝组织HBV DNA拷贝数仍可达到104~105;树鼩血清中HBV DNA拷贝数持续一个月高于正常组;肝组织炎细胞略增多,血清ALT水平持续升高.结论 rAAV8所携带的HBV基因组高效专一导入树鼩肝细胞并复制表达,成功建立HBV急性感染树鼩模型,为进一步探索rAAV8树鼩慢性感染模型打下一定的基础.%Objective To establish a tree shrew model of acute hepatitis B virus infection by injection of a recombinant adeno-associated virus 8 vector carrying 1.3 copies of HBV genome (ayw subtype) (rAAV8-1.3 HBV)into the liver of tree shrews.Methods Serum and liver tissues were collected at indicated times after i.v.injection of rAAV8-1.3 HBV into the tree shrews.The HBsAg,BeAg,HBsAb,HBeAb,HBcAb,ALT and HBV virus load were examined by ELISA and real-time PCR,respectively.The expression of HBcAg and pathological changes in the liver were also observed after the rAAV8-1.3 HBV infection.Results Markers of serum HBV were all positive 2 weeks after and HBcAg-positive hepatocytes were even detected in the liver 55 days after rAAV8-1.3 HBV injection.The copies of HBV DNA in liver reached 104-105 at 55 days after rAAV8-1.3HBV injection.Serum HBV DNA could be detected for over one month.Mild pathological changes with elevated ALT were observed after rAAV8-1.3 HBV injection.Conclusions A tree shrew

  15. Composting for Avian Influenza Virus Elimination

    OpenAIRE

    Elving, Josefine; Emmoth, Eva; Albihn, Ann; Vinnerås, Björn; Ottoson, Jakob

    2012-01-01

    Effective sanitization is important in viral epizootic outbreaks to avoid further spread of the pathogen. This study examined thermal inactivation as a sanitizing treatment for manure inoculated with highly pathogenic avian influenza virus H7N1 and bacteriophages MS2 and ϕ6. Rapid inactivation of highly pathogenic avian influenza virus H7N1 was achieved at both mesophilic (35°C) and thermophilic (45 and 55°C) temperatures. Similar inactivation rates were observed for bacteriophage ϕ6, while b...

  16. Avian Influenza: Mixed Infections and Missing Viruses

    OpenAIRE

    Wentworth, David E.; Dugan, Vivien G.; Xudong Lin; Seth Schobel; Magdalena Plancarte; Kelly, Terra R.; Lindsay, LeAnn L.; Boyce, Walter M.

    2013-01-01

    A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined ...

  17. Avian influenza virus risk assessment in falconry

    OpenAIRE

    Lüschow Dörte; Lierz Peter; Jansen Andreas; Harder Timm; Hafez Hafez; Kohls Andrea; Schweiger Brunhilde; Lierz Michael

    2011-01-01

    Abstract Background There is a continuing threat of human infections with avian influenza viruses (AIV). In this regard falconers might be a potential risk group because they have close contact to their hunting birds (raptors such as falcons and hawks) as well as their avian prey such as gulls and ducks. Both (hunting birds and prey birds) seem to be highly susceptible to some AIV strains, especially H5N1. We therefore conducted a field study to investigate AIV infections in falconers, their ...

  18. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

    OpenAIRE

    Lai, Yi; Yue, Yongping; LIU, MINGJU; Ghosh, Arkasubhra; Engelhardt, John F.; Jeffrey S. Chamberlain; Duan, Dongsheng

    2005-01-01

    Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient ...

  19. Aerosolized avian influenza virus by laboratory manipulations

    OpenAIRE

    Li Zhiping; Li Jinsong; Zhang Yandong; Li Lin; Ma Limin; Li Dan; Gao Feng; Xia Zhiping

    2012-01-01

    Abstract Background Avian H5N1 influenza viruses present a challenge in the laboratory environment, as they are difficult to collect from the air due to their small size and relatively low concentration. In an effort to generate effective methods of H5N1 air removal and ensure the safety of laboratory personnel, this study was designed to investigate the characteristics of aerosolized H5N1 produced by laboratory manipulations during research studies. Results Normal laboratory procedures used ...

  20. Virulence of Avian Influenza A Viruses for Squirrel Monkeys

    Science.gov (United States)

    Murphy, Brian R.; Hinshaw, Virginia S.; Sly, D. Lewis; London, William T.; Hosier, Nanette T.; Wood, Frank T.; Webster, Robert G.; Chanock, Robert M.

    1982-01-01

    Ten serologically distinct avian influenza A viruses were administered to squirrel monkeys and hamsters to compare their replication and virulence with those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the 10 avian influenza A viruses exhibited a spectrum of replication and virulence. The levels of virus replication and clinical response were closely correlated. Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8), resembled the human virus in their level and duration of replication and in their virulence. At the other end of the spectrum, five avian viruses were restricted by 100- to 10,000-fold in replication in the upper and lower respiratory tract and were clearly attenuated compared with the human influenza virus. In hamsters, the 10 viruses exhibited a spectrum of replication in the nasal turbinates, ranging from viruses that replicated as efficiently as the human virus to those that were 8,000- fold restricted. Since several avian viruses were closely related serologically to human influenza viruses, studies were done to confirm the avian nature of these isolates. Each of the avian viruses plaqued efficiently at 42°C, a restrictive temperature for replication of human influenza A viruses. Avian strains that had replicated either very efficiently or very poorly in squirrel monkeys still grew to high titer in the intestinal tracts of ducks, a tropism characteristic of avian, but not mammalian, influenza viruses. These observations indicate that some avian influenza A viruses grow well and cause disease in a primate host, whereas other avian viruses are very restricted in this host. These findings also provide a basis for determining the gene or genes involved in the restriction of replication that is observed with the attenuated avian viruses. Application of such information may allow the preparation of reassortant viruses derived from a virulent human influenza virus and an attenuated avian virus for possible

  1. Proteasome Inhibition Is Partially Effective in Attenuating Pre-Existing Immunity against Recombinant Adeno-Associated Viral Vectors

    OpenAIRE

    Karman, Jozsef; Gumlaw, Nathan K; Zhang, Jinhua; Jiang, Ji-Lei; Cheng, Seng H.; Zhu, Yunxiang

    2012-01-01

    Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezom...

  2. Antibody Neutralization Poses a Barrier to Intravitreal Adeno-Associated Viral Vector Gene Delivery to Non-Human Primates

    OpenAIRE

    Kotterman, Melissa A.; Yin, Lu; Strazzeri, Jennifer M.; Flannery, John G; Merigan, William H.; Schaffer, David V

    2014-01-01

    Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that pre-existing immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizin...

  3. Long-term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using Adeno associated Viral Gene Therapy

    OpenAIRE

    Chandler, Randy J.; Venditti, Charles P

    2009-01-01

    Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating meta...

  4. Transmission of Avian Influenza A Viruses Between Animals and People

    Science.gov (United States)

    ... Newsletters Transmission of Avian Influenza A Viruses Between Animals and People Language: English Español Recommend on ... Compartir Influenza A viruses have infected many different animals, including ducks, chickens, pigs, whales, horses, and seals. ...

  5. A role for adeno-associated viral vectors in gene therapy

    Directory of Open Access Journals (Sweden)

    Renata dos Santos Coura

    2008-01-01

    Full Text Available Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, have led to marked progress in this field. Although the available vector systems can successfully transfer genes into cells, the ideal delivery vehicle has not been found. In this context, adeno-associated virus vectors (AAV are arising as a promising tool for a wide range of applications, due to a combination of characteristics such as lack of pathogenicity and immunogenicity, wide range of cell tropism and long-term gene expression. Since its isolation, the biological properties of the adeno-associated virus have been increasingly understood, improving our ability to manipulate and use it as a safe and efficient gene therapy vector of wide spectrum. In this work, we review the bases of gene therapy, main types of gene transfer systems and basic properties and use of AAV vectors.

  6. Avian Influenza Viruses in Water Birds, Africa 1

    OpenAIRE

    Gaidet, Nicolas; Dodman, Tim; Caron, Alexandre; Balança, Gilles; Desvaux, Stephanie; Goutard, Flavie; Cattoli, Giovanni; Lamarque, François; Hagemeijer, Ward; Monicat, François

    2007-01-01

    We report the first large-scale surveillance of avian influenza viruses in water birds conducted in Africa. This study shows evidence of avian influenza viruses in wild birds, both Eurasian and Afro-tropical species, in several major wetlands of Africa.

  7. THE MOLECULAR BIOLOGY OF AVIAN INFLUENZA VIRUS IN SHORT

    Science.gov (United States)

    Avian influenza virus (AIV) is an important pathogen of poultry as it can cause severe economic losses through disease, including respiratory signs and mortality, and effects on trade. Avian influenza virus is classified as type A influenza, which is a member of the orthomyxoviridae family. Charact...

  8. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Institute of Scientific and Technical Information of China (English)

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

  9. [Progress in microRNAs associated with major avian viruses].

    Science.gov (United States)

    Man, Chaolai; Mu, Weitao; Zhao, Dongxue; Chang, Yang

    2015-09-01

    Recently, avian viral diseases have become one of the main models to study mechanisms of viral infections and pathogenesis. The study of regulatory relationships and mechanisms between viruses and microRNAs has also become the focus. In this review, we briefly summarize the general situations of microRNAs encoded by avian herpesviruses. Also, we analyze the regulatory relationships between tumorigenicity of avian herpesviruses and microRNAs. Additionally, the possible applications for prevention and treatment of viral diseases (such as infectious bursal disease, avian influenza and avian leucosis) using the regulatory mechanisms of microRNAs are also discussed. PMID:26955707

  10. Low-pathogenic avian influenza viruses in wild house mice.

    Directory of Open Access Journals (Sweden)

    Susan A Shriner

    Full Text Available BACKGROUND: Avian influenza viruses are known to productively infect a number of mammal species, several of which are commonly found on or near poultry and gamebird farms. While control of rodent species is often used to limit avian influenza virus transmission within and among outbreak sites, few studies have investigated the potential role of these species in outbreak dynamics. METHODOLOGY/PRINCIPAL FINDINGS: We trapped and sampled synanthropic mammals on a gamebird farm in Idaho, USA that had recently experienced a low pathogenic avian influenza outbreak. Six of six house mice (Mus musculus caught on the outbreak farm were presumptively positive for antibodies to type A influenza. Consequently, we experimentally infected groups of naïve wild-caught house mice with five different low pathogenic avian influenza viruses that included three viruses derived from wild birds and two viruses derived from chickens. Virus replication was efficient in house mice inoculated with viruses derived from wild birds and more moderate for chicken-derived viruses. Mean titers (EID(50 equivalents/mL across all lung samples from seven days of sampling (three mice/day ranged from 10(3.89 (H3N6 to 10(5.06 (H4N6 for the wild bird viruses and 10(2.08 (H6N2 to 10(2.85 (H4N8 for the chicken-derived viruses. Interestingly, multiple regression models indicated differential replication between sexes, with significantly (p<0.05 higher concentrations of avian influenza RNA found in females compared with males. CONCLUSIONS/SIGNIFICANCE: Avian influenza viruses replicated efficiently in wild-caught house mice without adaptation, indicating mice may be a risk pathway for movement of avian influenza viruses on poultry and gamebird farms. Differential virus replication between males and females warrants further investigation to determine the generality of this result in avian influenza disease dynamics.

  11. Avian influenza virus and free-ranging wild birds

    Science.gov (United States)

    Dierauf, Leslie A.; Karesh, W.B.; Ip, Hon S.; Gilardi, K.V.; Fischer, John R.

    2006-01-01

    Recent media and news reports and other information implicate wild birds in the spread of highly pathogenic avian influenza in Asia and Eastern Europe. Although there is little information concerning highly pathogenic avian influenza viruses in wild birds, scientists have amassed a large amount of data on low-pathogenicity avian influenza viruses during decades of research with wild birds. This knowledge can provide sound guidance to veterinarians, public health professionals, the general public, government agencies, and other entities with concerns about avian influenza.

  12. Infection of Avian Pox Virus in Oriental Turtle-Doves

    Directory of Open Access Journals (Sweden)

    Kyung-Yeon Eo1, Young-Hoan Kim2, Kwang-Hyun Cho3, Jong-Sik Jang4, Tae-Hwan Kim5, Dongmi Kwak5 and Oh-Deog Kwon5*

    2011-10-01

    Full Text Available Three Oriental Turtle-doves (Streptopelia orientalis exhibiting lethargy, dyspnea, poor physical condition, and poor flight endurance, were rescued and referred to the Animal Health Center, Seoul Zoo, Korea. The doves had wart-like lesions on the legs and head. All of them died the following day after arrival, with the exception of one that survived for 6 days. Diphtheritic membranes on the tongue and oral mucosa were apparent at necropsy. Avian pox virus infection was suspected based on the proliferative skin lesions and oral diphtheritic lesions. Infection of the avian pox virus was confirmed by PCR using primers specific to the 4b core protein gene of avian pox virus. All cases were diagnosed with avian pox virus infection. This is believed to be the first description on natural infection of avian pox in Oriental Turtle-doves in Korea.

  13. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  14. Avian Influenza: Mixed Infections and Missing Viruses

    Directory of Open Access Journals (Sweden)

    David E. Wentworth

    2013-08-01

    Full Text Available A high prevalence and diversity of avian influenza (AI viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.

  15. Virulence of Avian Influenza A Viruses for Squirrel Monkeys

    OpenAIRE

    Murphy, Brian R.; Hinshaw, Virginia S.; Sly, D. Lewis; London, William T.; Hosier, Nanette T.; Wood, Frank T.; Webster, Robert G.; Chanock, Robert M.

    1982-01-01

    Ten serologically distinct avian influenza A viruses were administered to squirrel monkeys and hamsters to compare their replication and virulence with those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the 10 avian influenza A viruses exhibited a spectrum of replication and virulence. The levels of virus replication and clinical response were closely correlated. Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8), resembled the human virus in t...

  16. Replication of avian influenza A viruses in mammals.

    OpenAIRE

    Hinshaw, V S; Webster, R. G.; Easterday, B C; Bean, W J

    1981-01-01

    The recent appearance of an avian influenza A virus in seals suggests that viruses are transmitted from birds to mammals in nature. To examine this possibility, avian viruses of different antigenic subtypes were evaluated for their ability to replicate in three mammals-pigs, ferrets, and cats. In each of these mammals, avian strains replicated to high titers in the respiratory tract (10(5) to 10(7) 50% egg infective doses per ml of nasal wash), with peak titers at 2 to 4 days post-inoculation...

  17. Analysis of avian leukosis virus infections with an enzyme immunoassay.

    OpenAIRE

    Clark, D P; Ball, R F; Dougherty, R M

    1981-01-01

    An enzyme-linked immunosorbent assay (ELISA) for avian leukosis virus group-specific antigen was used to study infections with and shedding of avian leukosis virus in a commercial flock of chickens with a known high incidence of infection. Avian leukosis virus group-specific antigen was detected in serum or cloacal washings from 76% of a group of 100 61-week-old hens. With eggs collected during the next 3 weeks, antigen was detected in the albumen of 88% of the eggs from ELISA-positive hens a...

  18. 腺相关病毒介导重组血管抑素联合雷公藤红素对大鼠颅内C6胶质瘤的抗血管生成作用%Anti-angiogenesis effect of adeno-associated virus-mediated recombinant angiostatin combined with celastrol on intracranial C6 glioma in rats

    Institute of Scientific and Technical Information of China (English)

    王冠; 周洁; 冯珂珂; 田麒

    2011-01-01

    目的:腺相关病毒(adeno-associated virus,AAV)介导的重组血管抑素(angiostatin,AS)联合应用雷公藤红素( celastrol)治疗大鼠颅内C6胶质瘤,观察其对肿瘤体积、新生血管密度及肿瘤细胞凋亡的影响,探讨抗血管生成重组基因联合雷公藤红素对胶质瘤治疗的前景.方法:建立颅内原位荷C6脑胶质瘤大鼠模型,7d后随机分为4组,分别给予0.9%氯化钠溶液(作为对照)、AAV-AS、雷公藤红素及两者联合用药.每隔7d行头部强化MRI检查,计算肿瘤体积.于22 d后处死动物,检测AS蛋白表达、血管密度及肿瘤细胞凋亡情况.结果:联合治疗组及AAV-AS治疗组均检测到AS蛋白表达,证实基因转导成功.联合治疗组第22天时肿瘤体积、血管密度和凋亡指数均与对照组、雷公藤红素组及AAV-AS治疗组相比差异有统计学意义(P<0.05),联合治疗可以抑制肿瘤生长,降低新生血管密度,促进肿瘤细胞凋亡.结论:基因治疗联合雷公藤红素可通过抑制胶质瘤血管生成而抑制肿瘤生长;两者联合应用具有协同作用,可弥补两者单独应用的不足之处.%Objective: To examine the effects of therapeutic alliance of adeno-associated virus-mediated recombinant angiostatin (AAV-AS) combined with celastrol on tumor growth, microvessel density and apoptosis of intracranial glioma in rats, and to give a prospective of this therapeutic alliance. Methods: A rat intracranial C6 glioma model was established, and then the rats (n=40) were randomly assigned into four groups after 7 days, which were saline control group, AAV-AS group, celastrol group and therapeutic alliance group. The tumor growth was examined by magnetic resonance imaging (MRI) every 7 days, and the volume of tumor was calculated. The rats were killed after 22 days, and the expression of AS protein, the microvessel density and the apoptosis of tumor cells were detected. Results: The expression of AS protein was detectable in AAV

  19. Avian influenza viruses - new causative a gents of human infections

    Directory of Open Access Journals (Sweden)

    Hrnjaković-Cvjetković Ivana

    2006-01-01

    Full Text Available Introduction. Influenza A viruses can infect humans, some mammals and especially birds. Subtypes of human influenza A viruses: ACH1N1, ACH2N2 and A(H3N2 have caused pandemics. Avian influenza viruses vary owing to their 15 hemagglutinins (H and 9 neuraminidases (N. Human cases of avian influenza A In the Netherlands in 2003, there were 83 human cases of influenza A (H7N7. In 1997, 18 cases of H5N1 influenza A, of whom 6 died, were found among residents of Hong Kong. In 2004, 34 human cases (23 deaths were reported in Viet Nam and Thailand. H5N1 virus-infected patients presented with fever and respiratory symptoms. Complications included respiratory distress syndrome, renal failure, liver dysfunction and hematologic disorders. Since 1999, 7 cases of human influenza H9N2 infection have been identified in China and Hong Kong. The importance of human infection with avian influenza viruses. H5N1 virus can directly infect humans. Genetic reassortment of human and avian influenza viruses may occur in humans co infected with current human A(HIN1 or A(H3N2 subtypes and avian influenza viruses. The result would be a new influenza virus with pandemic potential. All genes of H5Nl viruses isolated from humans are of avian origin. Prevention and control. The reassortant virus containing H and N from avian and the remaining proteins from human influenza viruses will probably be used as a vaccine strain. The most important control measures are rapid destruction of all infected or exposed birds and rigorous disinfection of farms. Individuals exposed to suspected animals should receive prophylactic treatment with antivirals and annual vaccination. .

  20. Aerosolized avian influenza virus by laboratory manipulations

    Directory of Open Access Journals (Sweden)

    Li Zhiping

    2012-08-01

    Full Text Available Abstract Background Avian H5N1 influenza viruses present a challenge in the laboratory environment, as they are difficult to collect from the air due to their small size and relatively low concentration. In an effort to generate effective methods of H5N1 air removal and ensure the safety of laboratory personnel, this study was designed to investigate the characteristics of aerosolized H5N1 produced by laboratory manipulations during research studies. Results Normal laboratory procedures used to process the influenza virus were carried out independently and the amount of virus polluting the on-site atmosphere was measured. In particular, zootomy, grinding, centrifugation, pipetting, magnetic stirring, egg inoculation, and experimental zoogenetic infection were performed. In addition, common accidents associated with each process were simulated, including breaking glass containers, syringe injection of influenza virus solution, and rupturing of centrifuge tubes. A micro-cluster sampling ambient air pollution collection device was used to collect air samples. The collected viruses were tested for activity by measuring their ability to induce hemagglutination with chicken red blood cells and to propagate in chicken embryos after direct inoculation, the latter being detected by reverse-transcription PCR and HA test. The results showed that the air samples from the normal centrifugal group and the negative-control group were negative, while all other groups were positive for H5N1. Conclusions Our findings suggest that there are numerous sources of aerosols in laboratory operations involving H5N1. Thus, laboratory personnel should be aware of the exposure risk that accompanies routine procedures involved in H5N1 processing and take proactive measures to prevent accidental infection and decrease the risk of virus aerosol leakage beyond the laboratory.

  1. Practical aspects of vaccination of poultry against avian influenza virus

    Science.gov (United States)

    Although little has changed in vaccine technology for avian influenza virus (AIV) in the past 20 years, the approach to vaccination of poultry (chickens, turkeys and ducks) for avian influenza has evolved as highly pathogenic (HP) AIV has become endemic in several regions of the world. Vaccination f...

  2. Highly Pathogenic Avian Influenza Virus Infection in Feral Raccoons, Japan

    OpenAIRE

    Horimoto, Taisuke; Maeda, Ken; Murakami, Shin; Kiso, Maki; Iwatsuki-Horimoto, Kiyoko; SASHIKA, Mariko; Ito, Toshihiro; Suzuki, Kazuo; Yokoyama, Mayumi; Kawaoka, Yoshihiro

    2011-01-01

    Although raccoons (Procyon lotor) are susceptible to influenza viruses, highly pathogenic avian influenza virus (H5N1) infection in these animals has not been reported. We performed a serosurvey of apparently healthy feral raccoons in Japan and found specific antibodies to subtype H5N1 viruses. Feral raccoons may pose a risk to farms and public health.

  3. Pathobiology of avian influenza virus infections in wild birds

    Science.gov (United States)

    Individual avian Influenza (AI) viruses vary in their ability to produce infection, disease and death in different bird species. Based on the pathobiological features in chickens, AI viruses (AIV) are categorized as low pathogenicity (LPAI) or high pathogenicity (HPAI) viruses, and can be of any of...

  4. Prevention and Treatment of Avian Influenza A Viruses in People

    Science.gov (United States)

    ... this? Submit What's this? Submit Button Past Newsletters Prevention and Treatment of Avian Influenza A Viruses in ... Recommend on Facebook Tweet Share Compartir The Best Prevention is to Avoid Sources of Exposure Currently, the ...

  5. Biology of Adeno-Associated Viral Vectors in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Aravind Asokan

    2014-09-01

    Full Text Available Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS. In particular, Adeno-Associated Viruses (AAV have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic.

  6. Avian influenza virus risk assessment in falconry

    Directory of Open Access Journals (Sweden)

    Lüschow Dörte

    2011-04-01

    Full Text Available Abstract Background There is a continuing threat of human infections with avian influenza viruses (AIV. In this regard falconers might be a potential risk group because they have close contact to their hunting birds (raptors such as falcons and hawks as well as their avian prey such as gulls and ducks. Both (hunting birds and prey birds seem to be highly susceptible to some AIV strains, especially H5N1. We therefore conducted a field study to investigate AIV infections in falconers, their falconry birds as well as prey birds. Findings During 2 hunting seasons (2006/2007 and 2007/2008 falconers took tracheal and cloacal swabs from 1080 prey birds that were captured by their falconry birds (n = 54 in Germany. AIV-RNA of subtypes H6, H9, or H13 was detected in swabs of 4.1% of gulls (n = 74 and 3.8% of ducks (n = 53 using RT-PCR. The remaining 953 sampled prey birds and all falconry birds were negative. Blood samples of the falconry birds tested negative for AIV specific antibodies. Serum samples from all 43 falconers reacted positive in influenza A virus-specific ELISA, but remained negative using microneutralisation test against subtypes H5 and H7 and haemagglutination inhibition test against subtypes H6, H9 and H13. Conclusion Although we were able to detect AIV-RNA in samples from prey birds, the corresponding falconry birds and falconers did not become infected. Currently falconers do not seem to carry a high risk for getting infected with AIV through handling their falconry birds and their prey.

  7. Surveillance of wild birds for avian influenza virus

    OpenAIRE

    Hoye, B.; Munster, V.J.; Nishiura, H.M.; Klaassen, M.; Fouchier, R. A. M.

    2010-01-01

    Recent demand for increased understanding of avian infl uenza virus in its natural hosts, together with the development of high-throughput diagnostics, has heralded a new era in wildlife disease surveillance. However, survey design, sampling, and interpretation in the context of host populations still present major challenges. We critically reviewed current surveillance to distill a series of considerations pertinent to avian infl uenza virus surveillance in wild birds, including consideratio...

  8. Detecting emerging transmissibility of avian influenza virus in human households

    OpenAIRE

    van Boven, M.; Koopmans, M.; Du Ry van Beest Holle, M.; Meijer, Adam; Klinkenberg, D.; Donnelly, C. A.; Heesterbeek, J A P

    2007-01-01

    Accumulating infections of highly pathogenic H5N1 avian influenza in humans underlines the need to track the ability of these viruses to spread among humans. A human-transmissible avian influenza virus is expected to cause clusters of infections in humans living in close contact. Therefore, epidemiological analysis of infection clusters in human households is of key importance. Infection clusters may arise from transmission events from (i) the animal reservoir, (ii) humans who were infected b...

  9. The challenges of avian influenza virus: mechanism, epidemiology and control

    Institute of Scientific and Technical Information of China (English)

    George F. GAO; Pang-Chui SHAW

    2009-01-01

    @@ Early 2009, eight human infection cases of H5N1 highly pathogenic avian influenza (HPAI) virus, with 5 death cases, were reported in China. This again made the world alert on a possible pandemic worldwide, probably caused by avian-origin influenza virus. Again H5N1 is in the spotlight of the world, not only for the scientists but also for the ordinary people. How much do we know about this virus? Where will this virus go and where did it come? Can we avoid a possible pandemic of influenza? Will the human beings conquer this devastating agent? Obviously we can list more questions than we know the answers.

  10. Avian influenza in shorebirds: experimental infection of ruddy turnstones (Arenaria interpres) with avian influenza virus

    Science.gov (United States)

    Hall, Jeffrey S.; Krauss, Scott; Franson, J. Christian; TeSlaa, Joshua L.; Nashold, Sean W.; Stallknecht, David E.; Webby, Richard J.; Webster, Robert G.

    2013-01-01

    Background: Low pathogenic avian influenza viruses (LPAIV) have been reported in shorebirds, especially at Delaware Bay, USA, during spring migration. However, data on patterns of virus excretion, minimal infectious doses, and clinical outcome are lacking. The ruddy turnstone (Arenaria interpres) is the shorebird species with the highest prevalence of influenza virus at Delaware Bay. Objectives: The primary objective of this study was to experimentally assess the patterns of influenza virus excretion, minimal infectious doses, and clinical outcome in ruddy turnstones. Methods: We experimentally challenged ruddy turnstones using a common LPAIV shorebird isolate, an LPAIV waterfowl isolate, or a highly pathogenic H5N1 avian influenza virus. Cloacal and oral swabs and sera were analyzed from each bird. Results: Most ruddy turnstones had pre-existing antibodies to avian influenza virus, and many were infected at the time of capture. The infectious doses for each challenge virus were similar (103·6–104·16 EID50), regardless of exposure history. All infected birds excreted similar amounts of virus and showed no clinical signs of disease or mortality. Influenza A-specific antibodies remained detectable for at least 2 months after inoculation. Conclusions: These results provide a reference for interpretation of surveillance data, modeling, and predicting the risks of avian influenza transmission and movement in these important hosts.

  11. Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

    Directory of Open Access Journals (Sweden)

    Fussenegger Martin

    2007-11-01

    Full Text Available Abstract Background Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. Results We designed recombinant adeno-associated virus (rAAV vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7. Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. Conclusion Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as

  12. Generation of transforming viruses in cultures of chicken fibroblasts infected with an avian leukosis virus.

    OpenAIRE

    Stavnezer, E; Gerhard, D S; Binari, R C; Balazs, I.

    1981-01-01

    During serial passages of an avian leukosis virus (the transformation-defective, src deletion mutant of Bratislava 77 avian sarcoma virus, designated tdB77) in chicken embryo fibroblasts, viruses which transformed chicken embryo fibroblasts in vitro emerged. Chicken embryo fibroblasts infected with these viruses (SK770 and Sk780) had a distinctive morphology, formed foci in monolayer cultures, and grew independent of anchorage in semisolid agar. Bone marrow cells were not transformed by these...

  13. Next generation sequencing technologies: tool to study avian virus diversity.

    Science.gov (United States)

    Kapgate, S S; Barbuddhe, S B; Kumanan, K

    2015-03-01

    Increased globalisation, climatic changes and wildlife-livestock interface led to emergence of novel viral pathogens or zoonoses that have become serious concern to avian, animal and human health. High biodiversity and bird migration facilitate spread of the pathogen and provide reservoirs for emerging infectious diseases. Current classical diagnostic methods designed to be virus-specific or aim to be limited to group of viral agents, hinder identifying of novel viruses or viral variants. Recently developed approaches of next-generation sequencing (NGS) provide culture-independent methods that are useful for understanding viral diversity and discovery of novel virus, thereby enabling a better diagnosis and disease control. This review discusses the different possible steps of a NGS study utilizing sequence-independent amplification, high-throughput sequencing and bioinformatics approaches to identify novel avian viruses and their diversity. NGS lead to the identification of a wide range of new viruses such as picobirnavirus, picornavirus, orthoreovirus and avian gamma coronavirus associated with fulminating disease in guinea fowl and is also used in describing viral diversity among avian species. The review also briefly discusses areas of viral-host interaction and disease associated causalities with newly identified avian viruses. PMID:25790045

  14. Quantitative Risk Assessment of Avian Influenza Virus Infection via Water

    NARCIS (Netherlands)

    Schijven FJ; Teunis PFM; Roda Husman AM de; MGB

    2006-01-01

    Using literature data, daily infection risks of chickens and humans with H5N1 avian influenza virus (AIV) by drinking water consumption were estimated for the Netherlands. A highly infectious virus and less than 4 log10 drinking water treatment (reasonably inefficient) may lead to a high infection r

  15. DETECTION OF AVIAN INFLUENZA VIRUS USING AN INTERFEROMETRIC BIOSENSOR

    Science.gov (United States)

    An optical interferometric waveguide immunoassay for direct and label-less detection of avian influenza virus is described. The assay response is based on index of refraction changes that occur upon binding of virus particles to antigen (hemagglutinin) specific antibodies on the waveguide surface. ...

  16. Immunohistochemical staining of avian influenza virus in tissues

    Science.gov (United States)

    Immunohistochemical methods are commonly used for studying the pathogenesis of avian influenza (AI) virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and metho...

  17. Avian influenza viruses - new causative a gents of human infections

    OpenAIRE

    Hrnjaković-Cvjetković Ivana; Cvjetković Dejan; Jerant-Patić Vera; Milošević Vesna; Tadić-Radovanov Jelena; Kovačević Gordana

    2006-01-01

    Introduction. Influenza A viruses can infect humans, some mammals and especially birds. Subtypes of human influenza A viruses: ACH1N1), ACH2N2) and A(H3N2) have caused pandemics. Avian influenza viruses vary owing to their 15 hemagglutinins (H) and 9 neuraminidases (N). Human cases of avian influenza A In the Netherlands in 2003, there were 83 human cases of influenza A (H7N7). In 1997, 18 cases of H5N1 influenza A, of whom 6 died, were found among residents of Hong Kong. In 2004, 34 human ca...

  18. Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

    OpenAIRE

    Scull, Margaret A.; Gillim-Ross, Laura; Santos, Celia; Roberts, Kim L.; Bordonali, Elena; Subbarao, Kanta; Barclay, Wendy S.; Pickles, Raymond J.

    2009-01-01

    Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal ...

  19. Seroepidemiological Evidence of Avian Influenza A Virus Transmission to Pigs in Southern China

    OpenAIRE

    Su, Shuo; Qi, Wenbao; Chen, Jidang; Zhu, Wanjun; Huang, Zhen; Xie, Jiexiong; Zhang, Guihong

    2013-01-01

    Recently, three novel avian-origin swine influenza viruses (SIVs) were first isolated from pigs in Guangdong Province, southern China, yet little is known about the seroprevalence of avian influenza viruses among pigs in southern China. Here, we report for the first time the seroprevalence of avian H3, H4, and H6 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 influenza virus transmission to pigs in China.

  20. Within-host variation of avian influenza viruses

    OpenAIRE

    Iqbal, Munir; Xiao, Hiaxia; Baillie, Greg; Warry, Andrew; Essen, Steve C.; Londt, Brandon; Brookes, Sharon M; Brown, Ian H.; McCauley, John W.

    2009-01-01

    The emergence and spread of H5N1 avian influenza viruses from Asia through to Europe and Africa pose a significant animal disease problem and have raised concerns that the virus may pose a pandemic threat to humans. The epizootological factors that have influenced the wide distribution of the virus are complex, and the variety of viruses currently circulating reflects these factors. Sequence analysis of the virus genes sheds light on the H5N1 virus evolution during its emergence and spread, b...

  1. Detecting emerging transmissibility of avian influenza virus in human households.

    Directory of Open Access Journals (Sweden)

    Michiel van Boven

    2007-07-01

    Full Text Available Accumulating infections of highly pathogenic H5N1 avian influenza in humans underlines the need to track the ability of these viruses to spread among humans. A human-transmissible avian influenza virus is expected to cause clusters of infections in humans living in close contact. Therefore, epidemiological analysis of infection clusters in human households is of key importance. Infection clusters may arise from transmission events from (i the animal reservoir, (ii humans who were infected by animals (primary human-to-human transmission, or (iii humans who were infected by humans (secondary human-to-human transmission. Here we propose a method of analysing household infection data to detect changes in the transmissibility of avian influenza viruses in humans at an early stage. The method is applied to an outbreak of H7N7 avian influenza virus in The Netherlands that was the cause of more than 30 human-to-human transmission events. The analyses indicate that secondary human-to-human transmission is plausible for the Dutch household infection data. Based on the estimates of the within-household transmission parameters, we evaluate the effectiveness of antiviral prophylaxis, and conclude that it is unlikely that all household infections can be prevented with current antiviral drugs. We discuss the applicability of our method for the detection of emerging human-to-human transmission of avian influenza viruses in particular, and for the analysis of within-household infection data in general.

  2. Pathogenicity of highly pathogenic avian influenza virus in mammals

    OpenAIRE

    de Wit, Emmie; Kawaoka, Yoshihiro; de Jong, Menno; Fouchier, Ron

    2008-01-01

    textabstractIn recent years, there has been an increase in outbreaks of highly pathogenic avian influenza (HPAI) in poultry. Occasionally, these outbreaks have resulted in transmission of influenza viruses to humans and other mammals, with symptoms ranging from conjunctivitis to pneumonia and death. Here, the current knowledge of the determinants of pathogenicity of HPAI viruses in mammals is summarized. It is becoming apparent that common mechanisms exist across influenza A virus strains and...

  3. 重组8型腺相关病毒介导双荧光素酶基因在小鼠体内的表达%Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse

    Institute of Scientific and Technical Information of China (English)

    王刚; 尉迟捷; 董小岩; 田文洪; 吴小兵

    2012-01-01

    目的 利用共表达的分泌型荧光素酶Gluc(gaussia princeps luciferase)和非分泌型荧光素酶Fluc(firefly luciferase)研究重组8型腺相关病毒(recombinant adeno-associated virus type 8,rAAV8)介导的转基因在小鼠体内的表达特点.方法 制备携带双荧光素酶基因的重组8型腺相关病毒rAAV8-Gluc/Fluc,体外感染HEK293细胞并检测上清和胞内Gluc和Fluc活性;将不同剂量的rAAV8-Gluc/Fluc尾静脉注射或肌内注射至BALB/c小鼠,通过尾静脉采血检测Gluc活性,通过活体成像和裂解组织检测Fluc活性.结果 成功制备了rAAV8-Gluc/Fluc,可以有效感染HEK293细胞,同时分泌表达Gluc和胞内表达Fluc;尾静脉注射或肌内注射rAAV8-Gluc/Fluc至小鼠后,外周血Gluc活性均在注射后10 ~20 d达到高峰并稳定持续120 d以上,Gluc活性随注射剂量增加而增高;静脉注射rAAV8-Gluc/Fluc时Fluc主要在肝脏表达,在骨骼肌和心肌有少量表达,而肌内注射时Fluc既在肌内注射局部表达同时也在肝脏中表达.结论 本研究成功制备了携带双荧光素酶基因rAAV8-Gluc/Fluc,研究了其介导的转基因在小鼠体内的表达特点,为rAAV8的临床前应用打下基础.%Objective Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).Methods rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro.BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im).Then Gluc activities in blood were measured,the whole-body images for Fluc activities were performed and Fluc activities of tissue lysate were also detected.Results rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells.The Gluc was mainly detected in the culture media while the Fluc was mainly

  4. Molecular diagnostics of Avian influenza virus

    Directory of Open Access Journals (Sweden)

    Petrović Tamaš

    2006-01-01

    direct sequencing of the PCR product. The possibility of typization using molecular methods is based on the big difference at the amino acid and nucleotide levels between different HA subtypes (from 20- 74%, while the differences between strains of the same HA subtype are relatively small (0- 9%. The basic advantage in the detection and typization of influenza viruses using the RTPCR method is that it saves time. Namely, it can be performed directly from the samples taken in the field, and the result can be obtained within the same day, contrary to conventional methods that take 7 to 10 days. The obtained PCR product can also be sequenced immediately, which can provide an answer to the possible virulent potential of the isolate and its further spreading. The establishment of changes in the HA gene sequence can provide us with the information about the direction of the development of the genetic drift. The paper will describe in detail the possibilities for the implementation of molecular methods in diagnostics and typization, in fact, in the molecular epizootiology of avian influenza.

  5. Avian leukosis virus infection: analysis of viremia and DNA integration in susceptible and resistant chicken lines.

    OpenAIRE

    Baba, T W; Humphries, E H

    1984-01-01

    Avian leukosis viruses induce lymphoid leukosis, a lymphoma which develops within the bursa of Fabricius several months after virus infection. Chickens from the Hyline SC and FP lines are, respectively, susceptible and resistant to avian leukosis virus-induced lymphoid leukosis. We examined plasma and cellular DNA obtained from avian leukosis virus-infected chickens for the presence of viremia and integrated viral sequences to determine whether the extent of virus infection is comparable in i...

  6. The Irrationality of GOF Avian Influenza Virus Research

    OpenAIRE

    Wain-Hobson, Simon

    2014-01-01

    The last two and a half years have witnessed a curious debate in virology characterized by a remarkable lack of discussion. It goes by the misleading epithet “gain of function” (GOF) influenza virus research, or simply GOF. As will be seen, there is nothing good to be gained. The controversial experiments confer aerosol transmission on avian influenza virus strains that can infect humans, but which are not naturally transmitted between humans. Some of the newer strains are clearly highly path...

  7. Molecular cloning of avian myelocytomatosis virus (MC29) transforming sequences.

    OpenAIRE

    Lautenberger, J A; Schulz, R A; Garon, C F; Tsichlis, P N; Papas, T S

    1981-01-01

    Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and to characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by So...

  8. Highly pathogenic avian influenza virus among wild birds in Mongolia

    Science.gov (United States)

    The central Asian country of Mongolia supports large populations of migratory water birds that migrate across much of Asia where highly pathogenic avian influenza (HPAI) virus subtype H5N1 is endemic. This, together with the near absence of domestic poultry, makes Mongolia an ideal location to unde...

  9. Research update: Avian Disease and Oncology Laboratory avian tumor viruses

    Science.gov (United States)

    Genomics and Immunogenetics Marek’s disease (MD), a lymphoproliferative disease caused by the highly oncogenic herpesvirus Marek's disease virus (MDV), continues to be a major disease concern to the poultry industry. The fear of MD is further enhanced by unpredictable vaccine breaks that result in ...

  10. Research update: Avian Disease and Oncology Laboratory avian tumor viruses

    Science.gov (United States)

    Genomics and Immunogenetics Use of genomics to identify QTL, genes, and proteins associated with resistance to Marek’s disease. Marek’s disease (MD), a lymphoproliferative disease caused by the highly oncogenic herpesvirus Marek's disease virus (MDV), continues to be a major disease concern to the p...

  11. Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection.

    OpenAIRE

    Crittenden, L B; McMahon, S.; Halpern, M S; Fadly, A M

    1987-01-01

    We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it late...

  12. Systemic delivery of genes to striated muscles using adeno-associated viral vectors.

    Science.gov (United States)

    Gregorevic, Paul; Blankinship, Michael J; Allen, James M; Crawford, Robert W; Meuse, Leonard; Miller, Daniel G; Russell, David W; Chamberlain, Jeffrey S

    2004-08-01

    A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adeno-associated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals. PMID:15273747

  13. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  14. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

    Science.gov (United States)

    Senís, Elena; Fatouros, Chronis; Große, Stefanie; Wiedtke, Ellen; Niopek, Dominik; Mueller, Ann-Kristin; Börner, Kathleen; Grimm, Dirk

    2014-11-01

    Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans. PMID:25186301

  15. Screening for Recombinant Avian Leukosis Viruses in Cell Cultures Inoculated with Various Subgroups of Virus

    Science.gov (United States)

    Chicken embryo fibroblasts (CEFs) prepared from ADOL SPF embryos were co-infected with different concentration ratios of subgroups A, J and E avian leukosis virus (ALV). Inoculated cultures were screened for recombination among the ALV strains. Potential recombinant viruses were purified by limiting...

  16. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

    Science.gov (United States)

    Selenica, Maj-Linda B; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B; Nash, Kevin R; Morgan, Dave; Gordon, Marcia N; Lee, Daniel C

    2016-05-01

    Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body

  17. Infection of Avian Pox Virus in Oriental Turtle-Doves

    OpenAIRE

    Kyung-Yeon Eo1, Young-Hoan Kim2, Kwang-Hyun Cho3, Jong-Sik Jang4, Tae-Hwan Kim5, Dongmi Kwak5 and Oh-Deog Kwon5*

    2011-01-01

    Three Oriental Turtle-doves (Streptopelia orientalis) exhibiting lethargy, dyspnea, poor physical condition, and poor flight endurance, were rescued and referred to the Animal Health Center, Seoul Zoo, Korea. The doves had wart-like lesions on the legs and head. All of them died the following day after arrival, with the exception of one that survived for 6 days. Diphtheritic membranes on the tongue and oral mucosa were apparent at necropsy. Avian pox virus infection was suspected based on the...

  18. Is low pathogenic avian influenza virus virulent for wild waterbirds?

    OpenAIRE

    Kuiken, T

    2013-01-01

    Although low pathogenic avian influenza virus (LPAIV) is traditionally considered to have adapted to its wild waterbird host to become avirulent, recent studies have suggested that LPAIV infection might after all have clinical effects. Therefore, I reviewed the literature on LPAIV infections in wild waterbirds. The virulence of LPAIV was assessed in 17 studies on experimental infections and nine studies on natural infections. Reported evidence for virulence were reductions in return rate, fee...

  19. Immunological phenotype of lymphomas induced by avian leukosis viruses.

    OpenAIRE

    Chen, L. C.; S.A. Courtneidge; Bishop, J M

    1983-01-01

    The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that...

  20. Rapid induction of hypothyroidism by an avian leukosis virus.

    OpenAIRE

    Carter, J K; Smith, R. E.

    1983-01-01

    Infection of 10-day chicken embryos with an avian leukosis virus, RAV-7, resulted in hypothyroidism within 3 weeks posthatching. Histological examination of the thyroids from infected chickens showed an extensive infiltration of lymphoblastoid cells by 7 days posthatching. Areas resembling germinal centers were present in the thyroids of infected chickens by 3 weeks posthatching. Examination of circulating thyroid and pancreas hormones showed a significant reduction in T3 and T4 levels and a ...

  1. Susceptibility of cell lines to avian viruses

    Directory of Open Access Journals (Sweden)

    Simoni Isabela Cristina

    1999-01-01

    Full Text Available The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.

  2. 重组腺相关病毒转导人树突状细胞体外诱导抗肝癌免疫应答%Generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells with adeno-associated virus expressing α-fetoprotein

    Institute of Scientific and Technical Information of China (English)

    杜文贞; 于天霞

    2011-01-01

    Objective To investigate the generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells (DC)with recombinant adeno-associated virus expressing α-fetoprotein (rAAV-AFP). Methods Peripheral blood mononuclear cells were isolated from healthy volunteers. Adherent peripheral blood mononuclear cells were transduced with AAV-AFP and cultured in the presence of granulocyte macrophage colony stimulating factor and interleukin-4 to generate dendritic cells.MTS assay was used to measure the ability of DC transduced with AAV-AFP ( AAV-AFP + DC) to stimulate the proliferation of T cell. The phenotype and AFP protein expression of DC and the secretion of IFN (interferon)-γ and IL (interleukin)-4 by T cells were detected by flow cytometry. The killing efficacy of cytotoxic T lymphocytes (CTL) activated by AAV-AFP + DC against AFP positive hepatocellular carcinoma cell lines was detected by lactate dehydrogenase (LDH) release assay. Results AAV-AFP + DC expressed HLA Ⅰ (97. 12%), HLAⅡ (97.32%), CD80(38.94%), CD83(60.84%)and CD86(98. 14%). AFP was secreted by 81.2% of AAV-AFP + DC. And it could stimulate effectively the proliferation of T cell.19. 84% of CD4 + T cells and 18.65% of CD8 + T cells activated by AAV-AFP + DC produced IFN-γbut not IL-4 and showed distinct killing activities against AFP positive hepatocellular carcinoma cell lines HepG2 (56. 45% ) and BEL7402 (78. 84% ). Conclusion AAV-AFP + DC can elicit distinct antitumor responses against AFP positive hepatocellular carcinoma cell lines so as to provide a basis for further researches on the clinical application of AAV-AFP + DC in the treatment of hepatocellular carcinoma.%目的 探讨携带甲胎蛋白基因的重组腺相关病毒(rAAV-AFP)转导人树突状细胞(DC)体外诱导抗肝癌免疫应答.方法 分离健康志愿者外周血单核细胞,贴壁细胞转导rAAV-AFP后,在粒细胞巨噬细胞集落刺激因子(GMCSF)和白细胞介素4(IL-4)的联

  3. Transmission of highly pathogenic avian influenza H7 virus

    OpenAIRE

    Bos, M.E.H.

    2009-01-01

    Knowledge of the transmission of highly pathogenic avian influenza (HPAI) virus still has gaps, complicating epidemic control. A model was developed to back-calculate the day HPAI virus was introduced into a flock, based on within-flock mortality data of the Dutch HPAI H7N7 epidemic (2003). The method was based on a stochastic epidemic model in which birds move from being susceptible, latently infected and infectious, to death. Our results indicated that two weeks can elapse before a noticeab...

  4. Specificity of avian leukosis virus-induced hyperlipidemia.

    OpenAIRE

    Carter, J K; Smith, R. E.

    1984-01-01

    Rous-associated virus 7 (RAV-7) is a subgroup C avian leukosis virus which does not transform cells in vitro or carry an oncogene. When injected into 1-day-old hatched chicks, RAV-7 causes a low incidence of lymphoid leukosis after a latent period of several months. In contrast, infection of 10-day-old chicken embryos with RAV-7 leads to a disease syndrome characterized by stunting, obesity, atrophy of the bursa and the thymus, high triglyceride and cholesterol levels, reduced thyroxine level...

  5. Nonconserved tryptophan 38 of the cell surface receptor for subgroup J avian leukosis virus discriminates sensitive from resistant avian species

    OpenAIRE

    Kučerová, D. (Dana); Plachý, J; Reinišová, M. (Markéta); Šenigl, F. (Filip); Trejbalová, K. (Kateřina); Geryk, J. (Josef); Hejnar, J. (Jiří)

    2013-01-01

    Subgroup J avian leukosis virus (ALV-J) is unique among the avian sarcoma and leukosis viruses in using the multimembrane-spanning cell surface protein Na+/H+ exchanger type 1 (NHE1) as a receptor. The precise localization of amino acids critical for NHE1 receptor activity is key in understanding the virus-receptor interaction and potential interference with virus entry. Because no resistant chicken lines have been described until now, we compared the NHE1 amino acid sequences from permissive...

  6. Antigenic properties of avian hepatitis E virus capsid protein.

    Science.gov (United States)

    Zhao, Qin; Syed, Shahid Faraz; Zhou, En-Min

    2015-10-22

    Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail. PMID:26340899

  7. Homo- and Heterosubtypic Low Pathogenic Avian Influenza Exposure on H5N1 Highly Pathogenic Avian Influenza Virus Infection in Wood Ducks (Aix sponsa)

    OpenAIRE

    Costa, Taiana P.; Brown, Justin D.; Howerth, Elizabeth W.; Stallknecht, David E.; Swayne, David E.

    2011-01-01

    Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. I...

  8. Molecular biology of avian infectious laryngotracheitis virus

    OpenAIRE

    Fuchs, Walter; Veits, Jutta; Helferich, Dorothee; Granzow, Harald; Teifke, Jens; Mettenleiter, Thomas

    2007-01-01

    Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an economically important chicken disease, which results in delayed growth, reduced egg production, and also frequently in death of the animals. After acute infection of the upper respiratory tract, the virus can establish latency in the central nervous system, and subsequent reactivations can lead to infection of naive chickens. For prevention of ILT, conventionally attenuated live vaccines are available. However, t...

  9. Complete Genome Sequence of an Avian-Like H4N8 Swine Influenza Virus Discovered in Southern China

    OpenAIRE

    Su, Shuo; Qi, Wen-bao; Chen, Ji-dang; Cao, Nan; Zhu, Wan-jun; Yuan, Li-Guo; Wang, Heng; Zhang, Gui-hong

    2012-01-01

    We report here the complete genomic sequence of an avian-like H4N8 swine influenza virus containing an H5N1 avian influenza virus segment from swine in southern China. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the Asia lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4N8 influenza virus to domestic pigs under natural conditions.

  10. Highly pathogenic avian influenza viruses inhibit effective immune responses of human blood-derived macrophages

    OpenAIRE

    Friesenhagen, Judith; Boergeling, Yvonne; Hrincius, Eike; Ludwig, Stephan; Roth, Johannes; Viemann, Dorothee

    2012-01-01

    Human blood-derived macrophages are non-permissive for influenza virus propagation, and fail to elicit inflammatory and antiviral responses upon infection with high pathogenic avian influenza viruses.

  11. Highly Pathogenic Avian Influenza Viruses and Generation of Novel Reassortants, United States, 2014–2015

    Science.gov (United States)

    Lee, Dong-Hun; Bahl, Justin; Torchetti, Mia Kim; Killian, Mary Lea; Ip, Hon S.; DeLiberto, Thomas J.

    2016-01-01

    Asian highly pathogenic avian influenza A(H5N8) viruses spread into North America in 2014 during autumn bird migration. Complete genome sequencing and phylogenetic analysis of 32 H5 viruses identified novel H5N1, H5N2, and H5N8 viruses that emerged in late 2014 through reassortment with North American low-pathogenicity avian influenza viruses. PMID:27314845

  12. Highly Pathogenic Avian Influenza Viruses and Generation of Novel Reassortants, United States, 2014-2015.

    Science.gov (United States)

    Lee, Dong-Hun; Bahl, Justin; Torchetti, Mia Kim; Killian, Mary Lea; Ip, Hon S; DeLiberto, Thomas J; Swayne, David E

    2016-07-01

    Asian highly pathogenic avian influenza A(H5N8) viruses spread into North America in 2014 during autumn bird migration. Complete genome sequencing and phylogenetic analysis of 32 H5 viruses identified novel H5N1, H5N2, and H5N8 viruses that emerged in late 2014 through reassortment with North American low-pathogenicity avian influenza viruses. PMID:27314845

  13. Highly pathogenic avian influenza viruses and generation of novel reassortants,United States, 2014–2015

    Science.gov (United States)

    Dong-Hun Lee; Justin Bahl; Mia Kim Torchetti; Mary Lea Killian; Ip, Hon S.; David E Swayne

    2016-01-01

    Asian highly pathogenic avian influenza A(H5N8) viruses spread into North America in 2014 during autumn bird migration. Complete genome sequencing and phylogenetic analysis of 32 H5 viruses identified novel H5N1, H5N2, and H5N8 viruses that emerged in late 2014 through reassortment with North American low-pathogenicity avian influenza viruses.

  14. Modelling the innate immune response against avian influenza virus in chicken

    NARCIS (Netherlands)

    Hagenaars, T.J.; Fischer, E.A.J.; Jansen, C.A.; Rebel, J.M.J.; Spekreijse, D.; Vervelde, L.; Backer, J.A.; Jong, de M.C.M.; Koets, A.P.

    2016-01-01

    At present there is limited understanding of the host immune response to (low pathogenic) avian influenza virus infections in poultry. Here we develop a mathematical model for the innate immune response to avian influenza virus in chicken lung, describing the dynamics of viral load, interferon-α,

  15. Evaluation and optimization of avian embryos and cell culture methods for efficient isolation and propagation of avian influenza viruses

    Science.gov (United States)

    Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting A...

  16. Zoonosis Update on H9N2 Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Abdul Ahad*, Masood Rabbani, Altaf Mahmood1, Zulfiqar Hussan Kuthu2, Arfan Ahmad and Muhammad Mahmudur Rahman3

    2013-07-01

    Full Text Available Influenza A viruses infect various mammals like human, horse, pig and birds as well. A total of 16 hemagglutinin (HA and 9 neuraminidase (NA subtypes have been identified. Most of the combinations are found in birds and relatively few have been isolated from mammals. Although there is no report of human to human transmission till to date, several cases of H5N1, H7N7 and H9N2 identified in humans since 1997 raised serious concern for health and veterinary profession. This review paper will focus H9N2 avian influenza virus (AIV with special emphasis on zoonosis. The virus H9N2 though not highly pathogenic like H5N1 but can be virulent through antigenic drift and shift.

  17. Molecular diagnostics of Avian influenza virus

    OpenAIRE

    Petrović Tamaš; Lazić Sava; Kapetanov Miloš; Velhner Maja

    2006-01-01

    The success of supervizing an infectious disease depends on the ability for speedy detection and characterization of the cause and the forming of a corresponding system for examining the success of control implemented in order to prevent a recurrence of the disease. Since influenza viruses continue to circle, causing significant morbidity and mortality both among the human population and among animals all over the world, it is essential to secure the timely identification and monitoring of th...

  18. Transmission dynamics of Avian Influenza A virus

    OpenAIRE

    Lu, Lu

    2015-01-01

    Influenza A virus (AIV) has an extremely high rate of mutation. Frequent exchanges of gene segments between different AIV (reassortment) have been responsible for major pandemics in recent human history. The presence of a wild bird reservoir maintains the threat of incursion of AIV into domestic birds, humans and other animals. In this thesis, I addressed unanswered questions of how diverse AIV subtypes (classified according to antigenicity of the two surface proteins, haema...

  19. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    Directory of Open Access Journals (Sweden)

    Mark W. Jackwood

    2011-09-01

    Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

  20. Subtype Identification of Avian Influenza Virus on DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    WANG Xiu-rong; YU Kang-zhen; DENG Guo-hua; SHI Rui; LIU Li-ling; QIAO Chuan-ling; BAO Hong-mei; KONG Xian-gang; CHEN Hua-lan

    2005-01-01

    We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus (AIV). The strains used in the experiment were A/Goose/Guangdong/1/96 (H5N1), A/African starling/983/79 (H7N1) and A/Turkey/Wiscosin/1/66 (H9N2). The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-1abeled fluorescent cDNAs,which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

  1. Lack of evidence of endogenous avian leukosis virus and endogenous avian retrovirus transmission to measles, mumps, and rubella vaccine recipients.

    OpenAIRE

    Hussain, A. I.; V. Shanmugam; Switzer, W. M.; Tsang, S. X.; Fadly, A.; Thea, D.; Helfand, R; Bellini, W J; Folks, T M; Heneine, W

    2001-01-01

    The identification of endogenous avian leukosis virus (ALV) and endogenous avian retrovirus (EAV) in chick cell-derived measles and mumps vaccines in current use has raised concern about transmission of these retroviruses to vaccine recipients. We used serologic and molecular methods to analyze specimens from 206 recipients of measles, mumps, and rubella (MMR) vaccine for evidence of infection with ALV and EAV. A Western blot assay for detecting antibodies to endogenous ALV was developed and ...

  2. E Protein Prokaryotic Expression of Avian Infectious Bronchitis Virus

    Institute of Scientific and Technical Information of China (English)

    WEI Ping; ZHANG Fang; MING Xiaobo; ZENG Xiangwei; ZHU Yuqing; WANG Lin

    2008-01-01

    The small envelope protein (E) gene of avian infectious bronchitis virus (IBV) M41 strain was cloned,and then it was subeloned into prokaryotic expressing vector pGEX-6P-1.The recombinant plasmid was transformed into E.coli.BL21 and induced by IPTG.SDS-PAGE result showed that when objective protein fused with GST (about 20 ku), the relative molecular mass of fusion protein was 38 ku.It indicated that objective protein was about 12.4 ku.The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research.

  3. Avian Influenza Viruses, Inflammation, and CD8+ T Cell Immunity

    OpenAIRE

    Wang, Zhongfang; Loh, Liyen; Kedzierski, Lukasz; Kedzierska, Katherine

    2016-01-01

    Avian influenza viruses (AIVs) circulate naturally in wild aquatic birds, infect domestic poultry, and are capable of causing sporadic bird-to-human transmissions. AIVs capable of infecting humans include a highly pathogenic AIV H5N1, first detected in humans in 1997, and a low pathogenic AIV H7N9, reported in humans in 2013. Both H5N1 and H7N9 cause severe influenza disease in humans, manifested by acute respiratory distress syndrome, multi-organ failure, and high mortality rates of 60% and ...

  4. Avian sarcoma virus 17 carries the jun oncogene.

    OpenAIRE

    Maki, Y; Bos, T J; Davis, C; Starbuck, M; Vogt, P K

    1987-01-01

    Biologically active molecular clones of avian sarcoma virus 17 (ASV 17) contain a replication-defective proviral genome of 3.5 kilobases (kb). The genome retains partial gag and env sequences, which flank a cell-derived putative oncogene of 0.93 kb, termed jun. The jun gene lacks preserved coding domains of tyrosine-specific protein kinases. It also shows no significant nucleic acid homology with other known oncogenes. The probable transformation-specific protein in ASV 17-transformed cells i...

  5. First characterization of avian influenza viruses from Greenland 2014

    DEFF Research Database (Denmark)

    Hartby, Christina Marie; Krog, Jesper Schak; Ravn Merkel, Flemming;

    2016-01-01

    In late February 2014, unusually high numbers of wild birds, thick-billed murre (Uria lomvia), were found dead at the coast of South Greenland. To investigate the cause of death, 45 birds were submitted for laboratory examinations in Denmark. Avian influenza viruses (AIVs) with subtypes H11N2 and...... emaciated appearance of birds, suggests that the murre die-off was not due to infection with AIV, but could be the mere cause of sparse food availability or stormy weather. Here we present the first characterization of AIVs isolated in Greenland, and our results support the idea that wild birds in Greenland...

  6. Transmission of Avian Influenza Virus (H3N2) to Dogs

    OpenAIRE

    Song, Daesub; Kang, Bokyu; Lee, Chulseung; Jung, Kwonil; Ha, Gunwoo; Kang, Dongseok; Park, Seongjun; Park, Bongkyun; Oh, Jinsik

    2008-01-01

    In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) is...

  7. Avian infectious bronchitis virus in Africa: a review.

    Science.gov (United States)

    Khataby, Khadija; Fellahi, Siham; Loutfi, Chafiqa; Mustapha, Ennaji Moulay

    2016-06-01

    Infectious bronchitis virus (IBV) is worldwide in distribution, highly infectious, and extremely difficult to control because it has extensive genetic diversity, a short generation time, and a high mutation rate. IBV is a Gammacoronavirus, single-stranded, and positive-sense RNA virus. Avian infectious bronchitis is well studied in European countries with identification of a large number of IBV variants, whereas in African countries epidemiological and scientific data are poor and not updated. However, previous studies reported that an IBV variant continues to appear regularly in Africa, as currently described in Morocco. No cross-protection between IBV strains was reported, some being unique to a particular country, others having a more general distribution. This review aims to provide a general overview on IB disease distribution in African countries and an update on the available studies of IBV variants in each country. PMID:27150555

  8. Avian myeloblastosis virus-induced lymphosarcoma producing erythroblastic leucosis in chicks

    Directory of Open Access Journals (Sweden)

    Kanzaki,Yoshito

    1975-10-01

    Full Text Available Acute myeloblastosis and several forms of tumor, including one case of lymphosarcoma occurred when avian myeloblastosis virus (BAI-A strain was inoculated into newly hatched chicks (SPF. The homogenate of lymphosarcoma inoculated intraperitoneally into other newly hatched chicks induced a high incidence of erythroblastic leucosis. Electron microscopy did not reveal the presence of C-type virus particles in the tumor tissue. The relationship between avian myeloblastosis virus, lymphosarcoma and erythroblastic leucosis is discussed.

  9. Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

    OpenAIRE

    Simon, M C; Neckameyer, W S; Hayward, W S; Smith, R. E.

    1987-01-01

    Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction o...

  10. Avian Influenza A Virus in Wild Birds in Highly Urbanized Areas

    OpenAIRE

    2012-01-01

    Avian influenza virus (AIV) surveillance studies in wild birds are usually conducted in rural areas and nature reserves. Less is known of avian influenza virus prevalence in wild birds located in densely populated urban areas, while these birds are more likely to be in close contact with humans. Influenza virus prevalence was investigated in 6059 wild birds sampled in cities in the Netherlands between 2006 and 2009, and compared with parallel AIV surveillance data from low urbanized areas in ...

  11. Surveillance of avian influenza viruses in Papua New Guinean poultry, June 2011 to April 2012

    OpenAIRE

    Marinjho Jonduo; Sook-San Wong; Nime Kapo; Paskalis Ominipi; Mohammad Abdad; Peter Siba; Pamela McKenzie; Richard Webby; Paul Horwood

    2013-01-01

    We investigated the circulation of avian influenza viruses in poultry populations throughout Papua New Guinea to assess the risk to the poultry industry and human health. Oropharyngeal swabs, cloacal swabs and serum were collected from 537 poultry from 14 provinces of Papua New Guinea over an 11–month period (June 2011 through April 2012). Virological and serological investigations were undertaken to determine the prevalence of avian influenza viruses. Neither influenza A viruses nor antibodi...

  12. Trans-Splicing Adeno-Associated Viral Vector-Mediated Gene Therapy Is Limited by the Accumulation of Spliced mRNA but Not by Dual Vector Coinfection Efficiency

    OpenAIRE

    XU, ZHUPING; Yue, Yongping; Lai, Yi; Ye, Chaoyang; Qiu, Jianming; Pintel, David J.; Duan, Dongsheng

    2004-01-01

    Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model ...

  13. Transmission and reassortment of avian influenza viruses at the Asian-North American interface

    Science.gov (United States)

    Ramey, Andrew M.; Pearce, John M.; Ely, Craig R.; Guy, Lisa M. Sheffield; Irons, David B.; Derksen, Dirk V.; Ip, Hon S.

    2010-01-01

    Twenty avian influenza viruses were isolated from seven wild migratory bird species sampled at St. Lawrence Island, Alaska. We tested predictions based on previous phylogenetic analyses of avian influenza viruses that support spatially dependent trans-hemispheric gene flow and frequent interspecies transmission at a location situated at the Asian–North American interface. Through the application of phylogenetic and genotypic approaches, our data support functional dilution by distance of trans-hemispheric reassortants and interspecific virus transmission. Our study confirms infection of divergent avian taxa with nearly identical avian influenza strains in the wild. Findings also suggest that H16N3 viruses may contain gene segments with unique phylogenetic positions and that further investigation of how host specificity may impact transmission of H13 and H16 viruses is warranted.

  14. Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation

    OpenAIRE

    Pengxiang Chang; Kuchipudi, Suresh V; Kenneth H. Mellits; Sujith Sebastian; Joe James; Jinhua Liu; Holly Shelton; Kin-Chow Chang

    2015-01-01

    Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) in...

  15. Methods to detect avian inlfuenza virus for food safety surveillance

    Institute of Scientific and Technical Information of China (English)

    SHI Ping; Shu Geng; LI Ting-ting; LI Yu-shui; FENG Ting; WU Hua-nan

    2015-01-01

    Avian inlfuenza (AI), caused by the inlfuenza A virus, has been a global concern for public health. AI outbreaks not only impact the poultry production, but also give rise to a risk in food safety caused by viral contamination of poultry products in the food supply chain. Distinctions in AI outbreak between strains H5N1 and H7N9 indicate that early detection of the AI virus in poultry is crucial for the effective warning and control of AI to ensure food safety. Therefore, the establishment of a poultry surveilance system for food safety by early detection is urgent and critical. In this article, methods to detect AI virus, including current methods recommended by the World Health Organization (WHO) and the World Organisation for Animal Health (Ofifce International des Epizooties, OIE) and novel techniques not commonly used or commercialized are reviewed and evaluated for feasibility of use in the poultry surveillance system. Conventional methods usualy applied for the purpose of AI diagnosis face some practical chalenges to establishing a comprehensive poultry surveilance program in the poultry supply chain. Diverse development of new technologies can meet the speciifc requirements of AI virus detec-tion in various stages or scenarios throughout the poultry supply chain where onsite, rapid and ultrasensitive methods are emphasized. Systematic approaches or integrated methods ought to be employed according to the application scenarios at every stage of the poultry supply chain to prevent AI outbreaks.

  16. Serological survey of avian influenza virus infection in non-avian wildlife in Xinjiang, China.

    Science.gov (United States)

    Wei, Yu-Rong; Yang, Xue-Yun; Li, Yuan-Guo; Wei, Jie; Ma, Wen-Ge; Ren, Zhi-Guang; Guo, Hui-Ling; Wang, Tie-Cheng; Mi, Xiao-Yun; Adili, Gulizhati; Miao, Shu-Kui; Shaha, Ayiqiaolifan; Gao, Yu-Wei; Huang, Jiong; Xia, Xian-Zhu

    2016-04-01

    We conducted a serological survey to detect antibodies against avian influenza virus (AIV) in Gazella subgutturosa, Canis lupus, Capreolus pygargus, Sus scrofa, Cervus elaphus, Capra ibex, Ovis ammon, Bos grunniens and Pseudois nayaur in Xinjiang, China. Two hundred forty-six sera collected from 2009 to 2013 were assayed for antibodies against H5, H7 and H9 AIVs using hemagglutination inhibition (HI) tests and a pan-influenza competitive ELISA. Across all tested wildlife species, 4.47 % harbored anti-AIV antibodies that were detected by the HI assay. The seroprevalence for each AIV subtype across all species evaluated was 0 % for H5 AIV, 0.81 % for H7 AIV, and 3.66 % for H9 AIV. H7-reactive antibodies were found in Canis lupus (9.09 %) and Ovis ammon (4.55 %). H9-reactive antibodies were found in Gazella subgutturosa (4.55 %), Canis lupus (27.27 %), Pseudois nayaur (23.08 %), and Ovis ammon (4.55 %). The pan-influenza competitive ELISA results closely corresponded to the cumulative prevalence of AIV exposure as measured by subtype-specific HI assays, suggesting that H7 and H9 AIV subtypes predominate in the wildlife species evaluated. These data provide evidence of prior infection with H7 and H9 AIVs in non-avian wildlife in Xinjiang, China. PMID:26733295

  17. A Complete Molecular Diagnostic Procedure for Applications in Surveillance and Subtyping of Avian Influenza Virus

    OpenAIRE

    Chun-Hsien Tseng; Hsiang-Jung Tsai; Chung-Ming Chang

    2014-01-01

    Introduction. The following complete molecular diagnostic procedure we developed, based on real-time quantitative PCR and traditional PCR, is effective for avian influenza surveillance, virus subtyping, and viral genome sequencing. Method. This study provides a specific and sensitive step-by-step procedure for efficient avian influenza identification of 16 hemagglutinin and 9 neuraminidase avian influenza subtypes. Result and Conclusion. This diagnostic procedure may prove exceedingly useful ...

  18. The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys.

    OpenAIRE

    Snyder, M H; Buckler-White, A J; London, W T; Tierney, E L; Murphy, B R

    1987-01-01

    Reassortant viruses which possessed the hemagglutinin and neuraminidase genes of wild-type human influenza A viruses and the remaining six RNA segments (internal genes) of the avian A/Pintail/Alberta/119/79 (H4N6) virus were previously found to be attenuated in humans. To study the genetic basis of this attenuation, we isolated influenza A/Pintail/79 X A/Washington/897/80 reassortant viruses which contained human influenza virus H3N2 surface glycoprotein genes and various combinations of avia...

  19. Influence of avian leukosis virus long terminal repeat on biological activities of Marek's disease virus.

    Science.gov (United States)

    Sun, Peng; Cui, Ning; Su, Shuai; Chen, Zimeng; Li, Yanpeng; Ding, Jiabo; Cui, Zhizhong

    2015-01-01

    GX0101 was the first reported field strain of recombinant Marek's disease virus (MDV) that contained a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). It is a very virulent MDV strain, with relatively high horizontal transmission ability. The REV LTR in GX0101 genome was proved to decrease the pathogenicity but increase the potential for horizontal transmission of the virus. Here we constructed a recombinant MDV GX0101-ALV-LTR to study stability of avian leukosis virus (ALV) LTR at the REV LTR insertion site in GX0101 genome and its influence on biological activities of the recombinant virus. The results showed that GX0101-ALV-LTR was able to replicate stably both in vitro and in vivo. ALV LTR remained stable in chickens infected either by inoculation with the recombinant virus GX0101-ALV-LTR or by horizontal transmission, as well as in cell culture. The pathogenic properties of GX0101-ALV-LTR virus were evaluated in infected specific-pathogen-free chickens. The present study demonstrated that the GX0101-ALV-LTR virus had a weaker inhibitory effect on the growth rates of the infected chickens and induced weaker immunosuppressive effects. Horizontal transmission ability of the GX0101-ALV-LTR virus appeared to be similar with its parental virus GX0101. In short, ALV LTR was stable in GX0101 after replacing REV LTR, and the recombinant virus showed similar horizontal transmission ability but decreased pathogenicity. PMID:26274570

  20. H5N1 Highly pathogenic avian influenza virus in wild birds

    Science.gov (United States)

    The existing H5N1 HPAI experimental infection data in wild avian species has validated observations made from field data and provided useful objective data on susceptibility, viral shedding, and pathobiology in different avian species. However, a complete understanding of the H5N1 HPAI virus epidem...

  1. Risk Perceptions for Avian Influenza Virus Infection among Poultry Workers, China

    OpenAIRE

    Yu, Qi; Liu, Linqing; Pu, Juan; Zhao, Jingyi; Sun, Yipeng; Shen, Guangnian; Wei, Haitao; Zhu, Junjie; Zheng, Ruifeng; Xiong, Dongyan; Liu, Xiaodong; Liu, Jinhua

    2013-01-01

    To determine risk for avian influenza virus infection, we conducted serologic surveillance for H5 and H9 subtypes among poultry workers in Beijing, China, 2009–2010, and assessed workers’ understanding of avian influenza. We found that poultry workers had considerable risk for infection with H9 subtypes. Increasing their knowledge could prevent future infections.

  2. Antibodies to H5 subtype avian influenza virus and Japanese encephalitis virus in northern pintails (Anas acuta) sampled in Japan

    Science.gov (United States)

    Blood samples from 105 northern pintails (Anas acuta) captured on Hokkaido, Japan were tested for antibodies to avian influenza virus (AIV), Japanese encephalitis virus (JEV) and West Nile virus (WNV) to assess possible involvement of this species in the transmission and spread of economically impor...

  3. Symmetrical base preferences surrounding HIV-1, avian sarcoma/leukosis virus, and murine leukemia virus integration sites

    OpenAIRE

    Holman, Alexander G; Coffin, John M.

    2005-01-01

    To investigate retroviral integration targeting on a nucleotide scale, we examined the base frequencies directly surrounding cloned in vivo HIV-1, murine leukemia virus, and avian sarcoma/leukosis virus integrations. Base preferences of up to 2-fold the expected frequencies were found for three viruses, representing P values down to

  4. Nonconserved tryptophan 38 of the cell surface receptor for subgroup J avian leukosis virus discriminates sensitive from resistant avian species

    Czech Academy of Sciences Publication Activity Database

    Kučerová, Dana; Plachý, Jiří; Reinišová, Markéta; Šenigl, Filip; Trejbalová, Kateřina; Geryk, Josef; Hejnar, Jiří

    2013-01-01

    Roč. 87, č. 15 (2013), s. 8399-8407. ISSN 0022-538X R&D Projects: GA ČR GAP502/10/1651 Institutional support: RVO:68378050 Keywords : avian leukosis virus * ALV-J * NHE1 * host resistance * receptor Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.648, year: 2013

  5. Inhibition of avian tumor viruses by vector-based RNA interference

    Science.gov (United States)

    RNA interference (RNAi) has been shown to reduce the replication of certain animal viruses both in cell culture and in live animals. We developed RNAi-based anti-viral strategies against two important chicken pathogens: avian leukosis virus (ALV) and Marek’s Disease virus MDV). Entry plasmids conta...

  6. Inactivation of avian influenza virus in chicken litter as a potential method to decontaminate poultry houses

    Science.gov (United States)

    Full cleaning and disinfection of a poultry house after an avian influenza virus (AIV) outbreak is expensive and labor intensive. An alternative to full house cleaning and disinfection is to inactivate the virus with high temperatures within the house. Litter in the house normally has a high virus...

  7. Subclinical Highly Pathogenic Avian Influenza Virus Infection among Vaccinated Chickens, China

    OpenAIRE

    Ma, Qing-Xia; Jiang, Wen-Ming; Liu, Shuo; Wang, Su-Chun; Zhuang, Qing-Ye; Hou, Guang-Yu; Liu, Xiang-Ming; Sui, Zheng-Hong; Chen, Ji-Ming

    2014-01-01

    Subclinical infection of vaccinated chickens with a highly pathogenic avian influenza A(H5N2) virus was identified through routine surveillance in China. Investigation suggested that the virus has evolved into multiple genotypes. To better control transmission of the virus, we recommend a strengthened program of education, biosecurity, rapid diagnostics, surveillance, and elimination of infected poultry.

  8. Novel Reassortant Highly Pathogenic Avian Influenza (H5N5) Viruses in Domestic Ducks, China

    OpenAIRE

    Gu, Min; Liu, Wenbo; Cao, Yongzhong; Peng, Daxin; Wang, Xiaobo; Wan, Hongquan; Zhao, Guo; Xu, Quangang; Zhang, Wei; Song, Qingqing; Li, Yanfang; Liu, Xiufan

    2011-01-01

    In China, domestic ducks and wild birds often share the same water, in which influenza viruses replicate preferentially. Isolation of 2 novel reassortant highly pathogenic avian influenza (H5N5) viruses from apparently healthy domestic ducks highlights the role of these ducks as reassortment vessels. Such new subtypes of influenza viruses may pose a pandemic threat.

  9. The challenges of avian influenza virus:mechanism,epidemiology and control

    Institute of Scientific and Technical Information of China (English)

    George; F.GAO; Pang-Chui; SHAW

    2009-01-01

    Early 2009, eight human infection cases of H5N1 highly pathogenic avian influenza (HPAI) virus, with 5 death cases, were reported in China. This again made the world alert on a possible pandemic worldwide, probably caused by

  10. Migratory birds reinforce local circulation of avian influenza viruses.

    Directory of Open Access Journals (Sweden)

    Josanne H Verhagen

    Full Text Available Migratory and resident hosts have been hypothesized to fulfil distinct roles in infectious disease dynamics. However, the contribution of resident and migratory hosts to wildlife infectious disease epidemiology, including that of low pathogenic avian influenza virus (LPAIV in wild birds, has largely remained unstudied. During an autumn H3 LPAIV epizootic in free-living mallards (Anas platyrhynchos - a partially migratory species - we identified resident and migratory host populations using stable hydrogen isotope analysis of flight feathers. We investigated the role of migratory and resident hosts separately in the introduction and maintenance of H3 LPAIV during the epizootic. To test this we analysed (i H3 virus kinship, (ii temporal patterns in H3 virus prevalence and shedding and (iii H3-specific antibody prevalence in relation to host migratory strategy. We demonstrate that the H3 LPAIV strain causing the epizootic most likely originated from a single introduction, followed by local clonal expansion. The H3 LPAIV strain was genetically unrelated to H3 LPAIV detected both before and after the epizootic at the study site. During the LPAIV epizootic, migratory mallards were more often infected with H3 LPAIV than residents. Low titres of H3-specific antibodies were detected in only a few residents and migrants. Our results suggest that in this LPAIV epizootic, a single H3 virus was present in resident mallards prior to arrival of migratory mallards followed by a period of virus amplification, importantly associated with the influx of migratory mallards. Thus migrants are suggested to act as local amplifiers rather than the often suggested role as vectors importing novel strains from afar. Our study exemplifies that a multifaceted interdisciplinary approach offers promising opportunities to elucidate the role of migratory and resident hosts in infectious disease dynamics in wildlife.

  11. Avian infectious bronchitis virus in Brazil: a highly complex virus meets a highly susceptible host population

    Directory of Open Access Journals (Sweden)

    PE Brandão

    2010-06-01

    Full Text Available Infectious bronchitis (IB is a highly aggressive disease for poultry in terms of symptoms and economic losses, and the control of this disease is difficult if flocks are not protected against type-specific challenges by the Avian infectious bronchitis virus (IBV. This article summarizes data presented by the author at the Workshop on Infectious Bronchitis 2009 on IB and IBV, including future developments on the field.

  12. Highly pathogenic avian influenza virus among wild birds in Mongolia.

    Directory of Open Access Journals (Sweden)

    Martin Gilbert

    Full Text Available Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005-2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured from 7,855 environmental fecal samples (primarily from ducks, or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans, while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2, or 2009 and 2010 (clade 2.3.2.1; all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study.

  13. Examination of presence of specific antibodies against avian influenza virus in some species of wild birds

    OpenAIRE

    Šekler Milanko; Ašanin Ružica; Krnjaić D.; Palić T.; Milić N.; Jovanović Tanja; Kovačević Dragana; Plavšić B.; Stojanović Dragica; Vidanović D.; Ašanin N.

    2009-01-01

    Infections caused by the avian influenza virus have been known for a long time and they are present, to a smaller or greater extent, in both extensive and intensive poultry production in many parts of the world. Epidemiological investigations have established a definite significance of the population of wild birds in maintaining and spreading this infection. Avian influenza is a zoonosis, and the virus has a great potential for causing mortality in humans, in particular its subtypes H5 and H7...

  14. Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs

    OpenAIRE

    Meng Qing-Wen; Zhang Zai-Ping; Wang Wei; Tian Jin; Xiao Zhi-Guang

    2011-01-01

    Abstract Background Avian leukosis virus (ALV) is a major infectious disease that impacts the poultry industry worldwide. Despite intensive efforts, no effective vaccine has been developed against ALV because of mutations that lead to resistant forms. Therefore, there is a dire need to develop antiviral agents for the treatment of ALV infections and RNA interference (RNAi) is considered an effective antiviral strategy. Results In this study, the avian leukosis virus subgroup J (ALV-J) provira...

  15. It is not just AIV: From avian to swine-origin influenza virus

    Institute of Scientific and Technical Information of China (English)

    GAO George F; SUN YePing

    2010-01-01

    @@ In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States.The virus spreads worldwide by human-to-human transmission.Within a few weeks, it reached a pandemic level.The virus is a novel reassorment virus.It contains gene fragments of influenza virus of swine, avian and human emerged from a triple reassortant virus circulating in North American swine.The source triple-reassortant itself comprised genes derived from avian (PB2 and PA), human H3N2 (PB1) and classical swine (HA, NP and NS) lineages.In contrast, the NA and M gene segments have their origin in the Eurasian avian-like swine H1N1 lineage (Figure 1).

  16. Development of an antigen-capture ELISA for the detection of avian leukosis virus p27 antigen.

    Science.gov (United States)

    Yun, Bingling; Li, Delong; Zhu, Haibo; Liu, Wen; Qin, Liting; Liu, Zaisi; Wu, Guan; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei; Gao, Yulong

    2013-02-01

    An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs. PMID:23201286

  17. Adeno-associated viral-mediated LARGE gene therapy rescues the muscular dystrophic phenotype in mouse models of dystroglycanopathy.

    Science.gov (United States)

    Yu, Miao; He, Yonglin; Wang, Kejian; Zhang, Peng; Zhang, Shengle; Hu, Huaiyu

    2013-03-01

    Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, and fukutin, creating the possibilities of a one-for-all gene therapy. To determine the feasibility of LARGE gene therapy, a serotype 9 adeno-associated viral vector for overexpressing LARGE (AAV9-LARGE) was injected intracardially into newborns of two mouse models of CMD: the natural LARGE mutant Large(myd) mice and protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice. AAV9-LARGE virus treatment yielded partial restoration of α-DG glycosylation and ligand-binding activity. The muscular dystrophy phenotype in skeletal muscles was ameliorated as revealed by significantly reduced fibrosis, necrosis, and numbers of centrally located nuclei with improved motor function. These results indicate that LARGE overexpression in vivo by AAV9-mediated gene therapy is effective at restoring functional glycosylation of α-DG and rescuing the muscular dystrophy phenotype in deficiency of not only LARGE but also POMGnT1, providing evidence that in vivo LARGE gene therapy may be broadly useful in dystroglycanopathies. PMID:23379513

  18. Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector.

    Science.gov (United States)

    Huang, Wei; McMurphy, Travis; Liu, Xianglan; Wang, Chuansong; Cao, Lei

    2016-06-01

    Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications. PMID:26857843

  19. Genome Sequence of a Novel Reassortant H3N2 Avian Influenza Virus in Southern China

    OpenAIRE

    Tian, Jin; Zhang, Changhui; Qi, Wenbao; XU, CHENGGANG; Huang, Lihong; Li, Huanan; Liao, Ming

    2012-01-01

    The distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a potential threat to both human and avian health. We report a complete genome sequence of a novel reassortant H3N2 avian influenza virus. Phylogenetic analysis showed that HA and NA showed the highest sequence homologies with those of A/white-backed munia/Hong Kong/4519/2009 (H3N2). However, the internal genes had the highest sequence homologies with those of H6 and H7 subtypes. The data ...

  20. Emergence of avian H1N1 influenza viruses in pigs in China.

    OpenAIRE

    Guan, Y.; Shortridge, K. F.; Krauss, S.; Li, P H; Kawaoka, Y.; Webster, R G

    1996-01-01

    Avian influenza A viruses from Asia are recognized as the source of genes that reassorted with human vital genes to generate the Asian/57 (H2N2) and Hong Kong/68 (H3N2) pandemic strains earlier in this century. Here we report the genetic analysis of avian influenza A H1N1 viruses recently isolated from pigs in southern China, a host suspected to generate new pandemic strains through gene reassortment events. Each of the eight gene segments was of avian origin. Phylogenetic analysis indicates ...

  1. Comparison of pathogenicities of H7 avian influenza viruses via intranasal and conjunctival inoculation in cynomolgus macaques.

    Science.gov (United States)

    Shichinohe, Shintaro; Itoh, Yasushi; Nakayama, Misako; Ozaki, Hiroichi; Soda, Kosuke; Ishigaki, Hirohito; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa

    2016-06-01

    The outbreak of H7N9 low pathogenic avian influenza viruses in China has attracted attention to H7 influenza virus infection in humans. Since we have shown that the pathogenicity of H1N1 and H5N1 influenza viruses in macaques was almost the same as that in humans, we compared the pathogenicities of H7 avian influenza viruses in cynomolgus macaques via intranasal and conjunctival inoculation, which mimics natural infection in humans. H7N9 virus, as well as H7N7 highly pathogenic avian influenza virus, showed more efficient replication and higher pathogenicity in macaques than did H7N1 and H7N3 highly pathogenic avian influenza viruses. These results are different from pathogenicity in chickens as reported previously. Therefore, our results obtained in macaques help to estimate the pathogenicity of H7 avian influenza viruses in humans. PMID:26994587

  2. Generation of a helper cell line for packaging avian leukosis virus-based vectors.

    OpenAIRE

    Savatier, P; Bagnis, C.; Thoraval, P; Poncet, D; Belakebi, M; Mallet, F.; Legras, C.; Cosset, F L; Thomas, J.L.; Chebloune, Y

    1989-01-01

    We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produ...

  3. Prevalence of Antibodies to H9N2 Avian Influenza Virus in Backyard Chickens around Maharlou Lake in Iran

    OpenAIRE

    Mohammad Mehdi Hadipour*, Gholamhossein Habibi and Amir Vosoughi

    2011-01-01

    Backyard chickens play an important role in the epidemiology of H9N2 avian influenza virus infection. Close contact of backyard chickens with migratory birds, especially with aquatic birds, as well as neighboring poultry farms, may pose the risk of transmitting avian influenza virus, but little is known about the disease status of backyard poultry. A H9N2 avian influenza virus seroprevalence survey was carried out in 500 backyard chickens from villages around Maharlou lake in Iran, using the ...

  4. Avian Influenza Viruses, Inflammation, and CD8+ T Cell Immunity

    Science.gov (United States)

    Wang, Zhongfang; Loh, Liyen; Kedzierski, Lukasz; Kedzierska, Katherine

    2016-01-01

    Avian influenza viruses (AIVs) circulate naturally in wild aquatic birds, infect domestic poultry, and are capable of causing sporadic bird-to-human transmissions. AIVs capable of infecting humans include a highly pathogenic AIV H5N1, first detected in humans in 1997, and a low pathogenic AIV H7N9, reported in humans in 2013. Both H5N1 and H7N9 cause severe influenza disease in humans, manifested by acute respiratory distress syndrome, multi-organ failure, and high mortality rates of 60% and 35%, respectively. Ongoing circulation of H5N1 and H7N9 viruses in wild birds and poultry, and their ability to infect humans emphasizes their epidemic and pandemic potential and poses a public health threat. It is, thus, imperative to understand the host immune responses to the AIVs so we can control severe influenza disease caused by H5N1 or H7N9 and rationally design new immunotherapies and vaccines. This review summarizes our current knowledge on AIV epidemiology, disease symptoms, inflammatory processes underlying the AIV infection in humans, and recent studies on universal pre-existing CD8+ T cell immunity to AIVs. Immune responses driving the host recovery from AIV infection in patients hospitalized with severe influenza disease are also discussed. PMID:26973644

  5. The Irrationality of GOF Avian Influenza Virus Research.

    Science.gov (United States)

    Wain-Hobson, Simon

    2014-01-01

    The last two and a half years have witnessed a curious debate in virology characterized by a remarkable lack of discussion. It goes by the misleading epithet "gain of function" (GOF) influenza virus research, or simply GOF. As will be seen, there is nothing good to be gained. The controversial experiments confer aerosol transmission on avian influenza virus strains that can infect humans, but which are not naturally transmitted between humans. Some of the newer strains are clearly highly pathogenic for man. It will be shown here that the benefits of the work are erroneous and overstated while the risk of an accident is finite, if small. The consequence of any accident would be anywhere from a handful of infections to a catastrophic pandemic. There has been a single open international meeting in this period, which is surprising given that openness and discussion are essential to good science. Despite US and EU government funding, no risk-benefit analysis has been published, which again is surprising. This research can be duplicated readily in many labs and requires little high tech. It falls under the definition of DURC without the slightest shadow of a doubt and constitutes the most important challenge facing contemporary biology. PMID:25077136

  6. First Characterization of Avian Influenza Viruses from Greenland 2014.

    Science.gov (United States)

    Hartby, Christina Marie; Krog, Jesper Schak; Merkel, Flemming; Holm, Elisabeth; Larsen, Lars Erik; Hjulsager, Charlotte Kristiane

    2016-05-01

    In late February 2014, unusually high numbers of wild thick-billed murres (Uria lomvia) were found dead on the coast of South Greenland. To investigate the cause of death, 45 birds were submitted for laboratory examination in Denmark. Avian influenza viruses (AIVs) with subtypes H11N2 and low pathogenic H5N1 were detected in some of the birds. Characterization of the viruses by full genome sequencing revealed that all the gene segments belonged to the North American lineage of AIVs. The seemingly sparse and mixed subtype occurrence of low pathogenic AIVs in these birds, in addition to the emaciated appearance of the birds, suggests that the murre die-off was due to malnutrition as a result of sparse food availability or inclement weather. Here we present the first characterization of AIVs isolated in Greenland, and our results support the idea that wild birds in Greenland may be involved in the movement of AIV between North America and Europe. PMID:27309071

  7. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    International Nuclear Information System (INIS)

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (Mpro), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV Mpro was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6122. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function

  8. Serological Evidence of Human Infection with Avian Influenza A H7virus in Egyptian Poultry Growers

    Science.gov (United States)

    Gomaa, Mokhtar R.; Kandeil, Ahmed; Kayed, Ahmed S.; Elabd, Mona A.; Zaki, Shaimaa A.; Abu Zeid, Dina; El Rifay, Amira S.; Mousa, Adel A.; Farag, Mohamed M.; McKenzie, Pamela P.; Webby, Richard J.; Ali, Mohamed A.; Kayali, Ghazi

    2016-01-01

    Avian influenza viruses circulate widely in birds, with occasional human infections. Poultry-exposed individuals are considered to be at high risk of infection with avian influenza viruses due to frequent exposure to poultry. Some avian H7 viruses have occasionally been found to infect humans. Seroprevalence of neutralizing antibodies against influenza A/H7N7 virus among poultry-exposed and unexposed individuals in Egypt were assessed during a three-years prospective cohort study. The seroprevalence of antibodies (titer, ≥80) among exposed individuals was 0%, 1.9%, and 2.1% annually while the seroprevalence among the control group remained 0% as measured by virus microneutralization assay. We then confirmed our results using western blot and immunofluorescence assays. Although human infection with H7 in Egypt has not been reported yet, our results suggested that Egyptian poultry growers are exposed to avian H7 viruses. These findings highlight the need for surveillance in the people exposed to poultry to monitor the risk of zoonotic transmission of avian influenza viruses. PMID:27258357

  9. Guinea pig model for evaluating the potential public health risk of swine and avian influenza viruses.

    Directory of Open Access Journals (Sweden)

    Yipeng Sun

    Full Text Available BACKGROUND: The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung. CONCLUSIONS/SIGNIFICANCE: We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.

  10. Diverse uses of feathers with emphasis on diagnosis of avian viral infections and vaccine virus monitoring

    Directory of Open Access Journals (Sweden)

    I Davidson

    2009-09-01

    Full Text Available The large amounts of feathers produced by the poultry industry, that is considered as a waste was explored for possible uses in various industries, such as meals for animals, biofuels, biodegradable plastic materials, combating water pollution and more. That review mentions these uses, but concentrate on the utilization of feathers for the diagnosis of viral infections and for monitoring vaccine viruses in chickens after vaccination. The viral diseases in which diagnosis using nucleic acids extracted from the feather shafts was described are, Marek's disease virus, circoviruses, chicken anemia virus, fowlpox virus, avian retroviruses, avian influenza virus and infectious laryngotracheitis virus. In two cases, of Marek's disease virus and of infectious laryngotracheitis virus, the differentiation of vaccine and wild-type viruses from feather shafts was made possible, thus allowing for monitoring the vaccination efficacy. The present review demonstrates also the stability of DNA viruses in feather shafts, and the possible evaluation of environmental dissemination of pathogens. When viruses are transmitted vertically, like in the cases of the retrovirus REV, a teratogenic effect on the development of feathers of the day-old newly hatched chick might occur in the case of avian influenza and the chicken anemia virus, which might indicate on a viral infection.

  11. Emergence of European Avian Influenza Virus-Like H1N1 Swine Influenza A Viruses in China▿

    OpenAIRE

    Liu, Jinhua; Bi, Yuhai; Qin, Kun; Fu, Guanghua; Yang, Jun; Peng, Jinshan; Ma, Guangpeng; Liu, Qinfang; Pu, Juan; Tian, Fulin

    2009-01-01

    During swine influenza surveillance from 2007 to 2008, 10 H1N1 viruses were isolated and analyzed for their antigenic and phylogenetic properties. Our study revealed the emergence of avian-origin European H1N1 swine influenza virus in China, which highlights the necessity of swine influenza surveillance for potential pandemic preparedness.

  12. Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses.

    Directory of Open Access Journals (Sweden)

    Darsaniya Punyadarsaniya

    Full Text Available BACKGROUND: Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7 was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. CONCLUSIONS/SIGNIFICANCE: Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones of swine.

  13. Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results.

    LENUS (Irish Health Repository)

    Flotte, Terence R

    2011-10-01

    Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes\\/kg (n=3 subjects\\/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg\\/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

  14. Experimental Infection of Dogs with Avian-Origin Canine Influenza A Virus (H3N2)

    OpenAIRE

    Song, Daesub; Lee, Chulseung; Kang, Bokyu; Jung, Kwonil; Oh, Taehoon; Kim, Hyekwon; Park, Bongkyun; Oh, Jinsik

    2009-01-01

    Susceptible dogs were brought into contact with dogs experimentally infected with an avian-origin influenza A virus (H3N2) that had been isolated from a pet dog with severe respiratory syndrome. All the experimentally infected and contact-exposed dogs showed elevated rectal temperatures, virus shedding, seroconversion, and severe necrotizing tracheobronchitis and bronchioalveolitis.

  15. Molecular-genetic analysis of field isolates of Avian Leucosis Viruses in the Russian Federation

    Science.gov (United States)

    Commercial poultry farms in 14 regions of Russian Federation were monitored for avian leukosis virus (ALV) infection using virus isolation tests and serology. Results indicated the presence of two subgroups of ALV in farms located in 11 of 14 regions. Analysis of the genomes of 12 field isolates of...

  16. New Avian Influenza Virus (H5N1) in Wild Birds, Qinghai, China

    OpenAIRE

    Li, Yanbing; Liu, Liling; Zhang, Yi; Duan, Zhenhua; Tian, Guobin; Zeng, Xianying; Shi, Jianzhong; Zhang, Licheng; Chen, Hualan

    2011-01-01

    Highly pathogenic avian influenza virus (H5N1) (QH09) was isolated from dead wild birds (3 species) in Qinghai, China, during May–June 2009. Phylogenetic and antigenic analyses showed that QH09 was clearly distinguishable from classical clade 2.2 viruses and belonged to clade 2.3.2.

  17. Highly Pathogenic Avian Influenza Virus A (H7N3) in Domestic Poultry, Saskatchewan, Canada, 2007

    OpenAIRE

    Berhane, Yohannes; Hisanaga, Tamiko; Kehler, Helen; Neufeld, James; Manning, Lisa; Argue, Connie; Handel, Katherine; Hooper-McGrevy, Kathleen; Jonas, Marilyn; Robinson, John; Webster, Robert G.; Pasick, John

    2009-01-01

    Epidemiologic, serologic, and molecular phylogenetic methods were used to investigate an outbreak of highly pathogenic avian influenza on a broiler breeding farm in Saskatchewan, Canada. Results, coupled with data from influenza A virus surveillance of migratory waterfowl in Canada, implicated wild birds as the most probable source of the low pathogenicity precursor virus.

  18. New Avian Influenza Virus (H5N1) in Wild Birds, Qinghai, China

    Science.gov (United States)

    Li, Yanbing; Liu, Liling; Zhang, Yi; Duan, Zhenhua; Tian, Guobin; Zeng, Xianying; Shi, Jianzhong; Zhang, Licheng

    2011-01-01

    Highly pathogenic avian influenza virus (H5N1) (QH09) was isolated from dead wild birds (3 species) in Qinghai, China, during May–June 2009. Phylogenetic and antigenic analyses showed that QH09 was clearly distinguishable from classical clade 2.2 viruses and belonged to clade 2.3.2. PMID:21291602

  19. Study of adeno-associated virus carrying the HGFK1 gene(AAV-HGFK1) in treating rat hepatocellular carcinoma%腺相关病毒介导的HGFK1对大鼠肝细胞癌的治疗作用研究

    Institute of Scientific and Technical Information of China (English)

    顾春荣; 郭跃武; 赵晖; 孙元珏; 姚阳; 沈赞; 林李家宓

    2009-01-01

    -angiogenesis molecule than angiostatin. In this study, we observed the effects and mechanisms of HGFK1 gene on the HCC. Methods: A recombinant adeno-associated vires carrying the HGFK1 gene (rAAV-HGFK1) was constructed.HCC of rat was induced by McA-RH7777. rAAV-HGFK1 was used to treat the rat, median survival time and metastasis rate were observed. Results: Ten days after tumor cell inoculation, surgery were performed to confirm the tumor formation, PBS, rAAV-EGFP or rAAV-HGFK1 was injected directly into the tumor nodule followed by portal vein injection. Results from our study demonstrated that rAAV-HGFK1 treatment significantly prolonged the median survival time of the HCC bearing rats from 30 days (PBS and rAAV-EGFP groups) to 49 days (rAAV-HGFK1 group). More importantly rAAV-HGFK1 inhibited tumor growth and completely prevented liver, lung and peritoneal metastasis. In the controlled PBS and AAV-EGFP group, liver and peritoneal metastasis rate were both 100%, and lung metastasis rate was 100% and 83%, respectively. While there was no metastasis found in treatment group, with only 33% of ascites happened. This was most possibly due to the primary tumor in liver but not due to the metastasis. Moreover, at a higher magnification (1000×), it was clear that the HGFK1 protein was expressed mainly in the cytoplasma of liver cells. In parallel, IHC staining of CD31 also demonstrated a significantly lower level of microvessel density (MVD) (6.21±1.6) in the liver tumor of the AAV-HGFK1 treatment group, as compared to the two control PBS and AAV-EGFP groups (25.1±2.1 and 26.8±2.5, respectively, P<0.01). HE staining showed that AAV-HGFK1 treatment induced large areas of necrosis in the tumor tissues, while minimal areas of necrosis were observed in the tumor tissue in the control groups. In addition, no toxicity appeared when high dosage (4.8× 1012 vg/rat) of rAAV-HGFK1 was administered in rats. Conclusion: Results from this study demonstrated that HGFK1 inhibited the growth and

  20. Role of Virus-Encoded microRNAs in Avian Viral Diseases

    OpenAIRE

    Yongxiu Yao; Venugopal Nair

    2014-01-01

    With total dependence on the host cell, several viruses have adopted strategies to modulate the host cellular environment, including the modulation of microRNA (miRNA) pathway through virus-encoded miRNAs. Several avian viruses, mostly herpesviruses, have been shown to encode a number of novel miRNAs. These include the highly oncogenic Marek’s disease virus-1 (26 miRNAs), avirulent Marek’s disease virus-2 (36 miRNAs), herpesvirus of turkeys (28 miRNAs), infectious laryngotracheitis virus (10 ...

  1. Effect of homosubtypic and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa)

    Science.gov (United States)

    Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus...

  2. Evaluation of a commercial blocking enzyme-linked immunosorbent assay to detect avian influenza virus antibodies in multiple experimentally infected avian species

    Science.gov (United States)

    Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoir for avian influenza (AI) viruses. Traditionally, AI surveillance in wild birds has relied on virus detection strategies including virus isolation and polymerase chain reaction. To evaluate the efficacy of a commerc...

  3. El virus influenza y la gripe aviar Influenza virus and avian flu

    Directory of Open Access Journals (Sweden)

    Libia Herrero-Uribe

    2008-03-01

    Full Text Available En este artículo se presenta una revisión del virus influenza,su biología,sus mecanismos de variación antigénica,las pandemias que ha producido y la prevención mediante las vacunas y medicamentos antivirales.Se analizan las razones por las cuales aparece el virus H5N1 que produce la fiebre aviar en humanos,la patogénesis de este virus y las estrategias para su prevención.Se informa sobre el plan de preparación para la pandemia en los niveles nacional e internacional.This article presents a review of Influenza virus,its biology,its mechanism of antigenic variation and its prevention by vaccination and the use of antivirals.The pandemics produced by this virus through history are presented.The appearance of the avian flu virus H5N1 is analyzed and its pathogenesis and strategies of prevention are discussed.National and international information about pandemic preparedness is presented.

  4. Avian Influenza A(H5N1) Virus in Egypt

    Science.gov (United States)

    Kandeil, Ahmed; El-Shesheny, Rabeh; Kayed, Ahmed S.; Maatouq, Asmaa M.; Cai, Zhipeng; McKenzie, Pamela P.; Webby, Richard J.; El Refaey, Samir; Kandeel, Amr; Ali, Mohamed A.

    2016-01-01

    In Egypt, avian influenza A subtype H5N1 and H9N2 viruses are enzootic in poultry. The control plan devised by veterinary authorities in Egypt to prevent infections in poultry focused mainly on vaccination and ultimately failed. Recently, widespread H5N1 infections in poultry and a substantial increase in the number of human cases of H5N1 infection were observed. We summarize surveillance data from 2009 through 2014 and show that avian influenza viruses are established in poultry in Egypt and are continuously evolving genetically and antigenically. We also discuss the epidemiology of human infection with avian influenza in Egypt and describe how the true burden of disease is underestimated. We discuss the failures of relying on vaccinating poultry as the sole intervention tool. We conclude by highlighting the key components that need to be included in a new strategy to control avian influenza infections in poultry and humans in Egypt. PMID:26886164

  5. Complete Genome Sequence of an H10N5 Avian Influenza Virus Isolated from Pigs in Central China

    OpenAIRE

    Wang, Nan; Zou, Wei; Yang, Ying; Guo, Xuebo; Hua, Yafeng; Qiang ZHANG; Zhao, Zongzheng; Jin, Meilin

    2012-01-01

    An avian H10N5 influenza virus, A/swine/Hubei/10/2008/H10N5, was isolated from pigs in the Hubei Province of central China. Homology and phylogenetic analyses of all eight gene segments demonstrated that the strain was wholly of avian origin and closely homologous to the Eurasian lineage avian influenza virus. To our knowledge, this is the first report of interspecies transmission of an avian H10N5 influenza virus to domestic pigs under natural conditions.

  6. Role of Virus-Encoded microRNAs in Avian Viral Diseases

    Science.gov (United States)

    Yao, Yongxiu; Nair, Venugopal

    2014-01-01

    With total dependence on the host cell, several viruses have adopted strategies to modulate the host cellular environment, including the modulation of microRNA (miRNA) pathway through virus-encoded miRNAs. Several avian viruses, mostly herpesviruses, have been shown to encode a number of novel miRNAs. These include the highly oncogenic Marek’s disease virus-1 (26 miRNAs), avirulent Marek’s disease virus-2 (36 miRNAs), herpesvirus of turkeys (28 miRNAs), infectious laryngotracheitis virus (10 miRNAs), duck enteritis virus (33 miRNAs) and avian leukosis virus (2 miRNAs). Despite the closer antigenic and phylogenetic relationship among some of the herpesviruses, miRNAs encoded by different viruses showed no sequence conservation, although locations of some of the miRNAs were conserved within the repeat regions of the genomes. However, some of the virus-encoded miRNAs showed significant sequence homology with host miRNAs demonstrating their ability to serve as functional orthologs. For example, mdv1-miR-M4-5p, a functional ortholog of gga-miR-155, is critical for the oncogenicity of Marek’s disease virus. Additionally, we also describe the potential association of the recently described avian leukosis virus subgroup J encoded E (XSR) miRNA in the induction of myeloid tumors in certain genetically-distinct chicken lines. In this review, we describe the advances in our understanding on the role of virus-encoded miRNAs in avian diseases. PMID:24662606

  7. Role of Virus-Encoded microRNAs in Avian Viral Diseases

    Directory of Open Access Journals (Sweden)

    Yongxiu Yao

    2014-03-01

    Full Text Available With total dependence on the host cell, several viruses have adopted strategies to modulate the host cellular environment, including the modulation of microRNA (miRNA pathway through virus-encoded miRNAs. Several avian viruses, mostly herpesviruses, have been shown to encode a number of novel miRNAs. These include the highly oncogenic Marek’s disease virus-1 (26 miRNAs, avirulent Marek’s disease virus-2 (36 miRNAs, herpesvirus of turkeys (28 miRNAs, infectious laryngotracheitis virus (10 miRNAs, duck enteritis virus (33 miRNAs and avian leukosis virus (2 miRNAs. Despite the closer antigenic and phylogenetic relationship among some of the herpesviruses, miRNAs encoded by different viruses showed no sequence conservation, although locations of some of the miRNAs were conserved within the repeat regions of the genomes. However, some of the virus-encoded miRNAs showed significant sequence homology with host miRNAs demonstrating their ability to serve as functional orthologs. For example, mdv1-miR-M4-5p, a functional ortholog of gga-miR-155, is critical for the oncogenicity of Marek’s disease virus. Additionally, we also describe the potential association of the recently described avian leukosis virus subgroup J encoded E (XSR miRNA in the induction of myeloid tumors in certain genetically-distinct chicken lines. In this review, we describe the advances in our understanding on the role of virus-encoded miRNAs in avian diseases.

  8. Role of virus-encoded microRNAs in Avian viral diseases.

    Science.gov (United States)

    Yao, Yongxiu; Nair, Venugopal

    2014-01-01

    With total dependence on the host cell, several viruses have adopted strategies to modulate the host cellular environment, including the modulation of microRNA (miRNA) pathway through virus-encoded miRNAs. Several avian viruses, mostly herpesviruses, have been shown to encode a number of novel miRNAs. These include the highly oncogenic Marek's disease virus-1 (26 miRNAs), avirulent Marek's disease virus-2 (36 miRNAs), herpesvirus of turkeys (28 miRNAs), infectious laryngotracheitis virus (10 miRNAs), duck enteritis virus (33 miRNAs) and avian leukosis virus (2 miRNAs). Despite the closer antigenic and phylogenetic relationship among some of the herpesviruses, miRNAs encoded by different viruses showed no sequence conservation, although locations of some of the miRNAs were conserved within the repeat regions of the genomes. However, some of the virus-encoded miRNAs showed significant sequence homology with host miRNAs demonstrating their ability to serve as functional orthologs. For example, mdv1-miR-M4-5p, a functional ortholog of gga-miR-155, is critical for the oncogenicity of Marek's disease virus. Additionally, we also describe the potential association of the recently described avian leukosis virus subgroup J encoded E (XSR) miRNA in the induction of myeloid tumors in certain genetically-distinct chicken lines. In this review, we describe the advances in our understanding on the role of virus-encoded miRNAs in avian diseases. PMID:24662606

  9. The Influence of Ecological Factors on the Transmission and Stability of Avian Influenza Virus in the Environment

    Directory of Open Access Journals (Sweden)

    Dyah Ayu Hewajuli

    2014-09-01

    Full Text Available Ecology is a science studying the correlation among organisms and some environmental factors. Ecological factors play an important role to transmit Avian Influenza (AI virus and influence its stability in the environment. Avian Influenza virus is classified as type A virus and belong to Orthomyxoviridae family. The virus can infect various vertebrates, mainly birds and mammals, including human. Avian Influenza virus transmission can occur through bird migration. The bird migration patterns usually occur in the large continent covers a long distance area within a certain periode hence transmit the virus from infected birds to other birds and spread to the environment. The biotic (normal flora microbes and abiotic (physical and chemical factors play important role in transmitting the virus to susceptible avian species and influence its stability in the environment. Disinfectant can inactivate the AI virus in the environment but its effectivity is influenced by the concentration, contact time, pH, temperature and organic matter.

  10. Genome characterisation of the newly discovered avian influenza A H5N7 virus subtype combination

    DEFF Research Database (Denmark)

    Bragstad, K.; Jørgensen, Poul Henrik; Handberg, K.J.; Fomsgaard, A.

    2007-01-01

    In Denmark, in 2003, a previously unknown subtype combination of avian influenza A virus, H5N7 (A/Mallard/Denmark/64650/03), was isolated from a flock of 12,000 mallards. The H5N7 subtype combination might be a reassortant between recent European avian influenza A H5, H7, and a third subtype....../Duck/Hong Kong/3096/99 (H6N2) and A/WDk/ST/1737/2000 (H6N8), respectively. All genes of the H5N7 strain were of avian origin, and no further evidence of pathogenicity to humans has been found....

  11. First detection of highly pathogenic avian influenza virus H5N1 in common kestrel falcon (Falco tinnunculus) in Egypt

    OpenAIRE

    ElBakrey, Reham M.; Mansour, Shimaa M. G.; Ali, Haytham; Knudsen, David E. B.; Eid, Amal A. M.

    2016-01-01

    Highly pathogenic avian influenza virus (HPAIV) poses threats to animal and human health worldwide. A common kestrel (Falco tinnunculus) was submitted to Avian and Rabbit Medicine Department, Zagazig University, Egypt. It exhibited torticollis, incoordination, and inability to stand. Conjunctivitis and crust formation were seen. Postmortem findings revealed congestion in internal organs and greenish content in gizzard. No avian pox virus was detected in cutaneous lesions neither in histopatho...

  12. The variable codons of H5N1 avian influenza A virus haemagglutinin genes

    Institute of Scientific and Technical Information of China (English)

    Mark; J.GIBBS; Robert; W.MURPHY

    2008-01-01

    We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsyn-onymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in vi-ruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected posi-tions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.

  13. Correlation between cell killing and massive second-round superinfection by members of some subgroups of avian leukosis virus.

    OpenAIRE

    Weller, S K; Joy, A E; Temin, H M

    1980-01-01

    Avian leukosis viruses of subgroups B, D, and F are cytopathic for chicken cells, whereas viruses of subgroups A, C, and E are not. The amounts of unintegrated linear viral DNA in cells at different times after infection with cytopathic or noncytopathic viruses were determined by hybridization and transfection assays. Shortly after infection, there is a transient accumulation of unintegrated linear viral DNA in cells infected with cytopathic avian leukosis viruses. By 10 days after infection,...

  14. Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

    OpenAIRE

    Cosset, F L; Legras, C.; Thomas, J.L.; Molina, R. M.; Chebloune, Y; Faure, C.; Nigon, V M; Verdier, G

    1991-01-01

    Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gav...

  15. Complete Genome Sequence of a Novel Avian-Like H3N2 Swine Influenza Virus Discovered in Southern China

    OpenAIRE

    Su, Shuo; Chen, Ji-dang; Qi, Hai-tao; Zhu, Wan-jun; Xie, Jie-xiong; Huang, Zhen; Tan, Li-kai; Qi, Wen-bao; Zhang, Gui-hong

    2012-01-01

    We report here the complete genomic sequence of a novel avian-like H3N2 swine influenza virus containing an H5N1 highly pathogenic avian influenza virus segment that was obtained from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic aquatic birds. The sequence information provided herein suggests that continuing study is required to determine if this virus can be established in the swine population and pose potential threats to public health.

  16. An emerging avian influenza A virus H5N7 is a genetic reassortant of highly pathogenic genes

    DEFF Research Database (Denmark)

    Bragstad, K.; Jørgensen, Poul Henrik; Handberg, Kurt;

    2006-01-01

    We full genome characterised the newly discovered avian influenza virus H5N7 subtype combination isolated from a stock of Danish game ducks to investigate the composition of the genome and possible features of high pathogenicity. It was found that the haemagglutinin and the acidic polymerase gene...... low pathogenic avian influenza A viruses. (c) 2006 Elsevier Ltd. All rights reserved....

  17. Replacement of primary chicken embryonic fibroblasts (CEF) by the DF-1 cell line for detection of avian leucosis viruses

    NARCIS (Netherlands)

    Maas, van der R.; Zoelen-Bos, van D.J.; Oei, H.L.; Claassen, I.J.T.M.

    2006-01-01

    International regulations prescribe that the absence of avian leucosis viruses (ALV) in avian live virus vaccines has to be demonstrated. Primary chicken embryo fibroblasts (CEF) from special SPF chicken lines are normally used for detection of ALV. The suitability of the DF-1 cell line for ALV-dete

  18. Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses

    DEFF Research Database (Denmark)

    Maines, Taronna R; Chen, Li-Mei; Van Hoeven, Neal; Tumpey, Terrence M; Blixt, Ola; Belser, Jessica A; Gustin, Kortney M; Pearce, Melissa B; Pappas, Claudia; Stevens, James; Cox, Nancy J; Paulson, James C; Raman, Rahul; Sasisekharan, Ram; Katz, Jacqueline M; Donis, Ruben O

    2011-01-01

    Although H5N1 influenza viruses have been responsible for hundreds of human infections, these avian influenza viruses have not fully adapted to the human host. The lack of sustained transmission in humans may be due, in part, to their avian-like receptor preference. Here, we have introduced...

  19. Antigenic Characterization of H3 Subtypes of Avian Influenza A Viruses from North America.

    Science.gov (United States)

    Bailey, Elizabeth; Long, Li-Ping; Zhao, Nan; Hall, Jeffrey S; Baroch, John A; Nolting, Jacqueline; Senter, Lucy; Cunningham, Frederick L; Pharr, G Todd; Hanson, Larry; Slemons, Richard; DeLiberto, Thomas J; Wan, Xiu-Feng

    2016-05-01

    Besides humans, H3 subtypes of influenza A viruses (IAVs) can infect various animal hosts, including avian, swine, equine, canine, and sea mammal species. These H3 viruses are both antigenically and genetically diverse. Here, we characterized the antigenic diversity of contemporary H3 avian IAVs recovered from migratory birds in North America. Hemagglutination inhibition (HI) assays were performed on 37 H3 isolates of avian IAVs recovered from 2007 to 2011 using generated reference chicken sera. These isolates were recovered from samples taken in the Atlantic, Mississippi, Central, and Pacific waterfowl migration flyways. Antisera to all the tested H3 isolates cross-reacted with each other and, to a lesser extent, with those to H3 canine and H3 equine IAVs. Antigenic cartography showed that the largest antigenic distance among the 37 avian IAVs is about four units, and each unit corresponds to a 2 log 2 difference in the HI titer. However, none of the tested H3 IAVs cross-reacted with ferret sera derived from contemporary swine and human IAVs. Our results showed that the H3 avian IAVs we tested lacked significant antigenic diversity, and these viruses were antigenically different from those circulating in swine and human populations. This suggests that H3 avian IAVs in North American waterfowl are antigenically relatively stable. PMID:27309078

  20. Antigenic Characterization of H3 Subtypes of Avian Influenza A Viruses from North America

    Science.gov (United States)

    Bailey, Elizabeth; Long, Li-Ping; Zhao, Nan; Hall, Jeffrey S.; Baroch, John A.; Nolting, Jacqueline; Senter, Lucy; Cunningham, Frederick L.; Pharr, G. Todd; Hanson, Larry; Slemons, Richard; DeLiberto, Thomas J.; Wan, Xiu-Feng

    2016-01-01

    SUMMARY Besides humans, H3 subtypes of influenza A viruses (IAVs) can infect various animal hosts including avian, swine, equine, canine, and sea mammals. These H3 viruses are both antigenically and genetically diverse. Here we characterized the antigenic diversity of contemporary H3 avian IAVs recovered from migratory birds in North America. Hemagglutination inhibition (HI) assays were performed on 37 H3 isolates of avian IAVs recovered from 2007 to 2011 using generated reference chicken sera. These isolates were recovered from samples taken in the Atlantic, Mississippi, Central, and Pacific waterfowl migration flyways. Antisera to all the tested H3 isolates cross-reacted with each other, and, to a lesser extent, with those to H3 canine and H3 equine IAVs. Antigenic cartography showed that the largest antigenic distance among the 37 avian IAVs is about 4 units, and each unit corresponds to a 2log2 difference in the HI titer. However, none of the tested H3 IAVs cross-reacted with ferret sera derived from contemporary swine and human IAVs. Our results showed that the H3 avian IAVs we tested lacked significant antigenic diversity, and these viruses were antigenically different from those circulating in swine and human populations. This suggests that H3 avian IAVs in North American waterfowl are antigenically relatively stable. PMID:27309078

  1. At least two regions of the viral genome determine the oncogenic potential of avian leukosis viruses.

    OpenAIRE

    Robinson, H L; Blais, B M; Tsichlis, P N; Coffin, J. M.

    1982-01-01

    Recombinants of oncogenic and nononcogenic avian leukosis viruses were tested for their oncogenic potential in chickens. The results indicate that at least two regions of the viral genome determine the oncogenic potential of these viruses. The first region contains sequences that control viral mRNA synthesis. These sequences determine the potential of a virus to induce a low incidence of lymphomas, carcinomas, chondrosarcomas, fibrosarcomas, and osteopetrosis. The second region lies outside t...

  2. Avian-origin H3N2 canine influenza A viruses in Southern China

    OpenAIRE

    Li, Shoujun; Shi, Zhihai; Jiao, Peirong; Zhang, Guihong; Zhong, Zhiwen; Tian, Wenru; Long, Li-Ping; Cai, Zhipeng; Zhu, Xingquan; Liao, Ming; Wan, Xiu-Feng

    2010-01-01

    This study reports four sporadic cases of H3N2 canine influenza in southern China, which were identified from sick dogs from May 2006 to October 2007. The evolutionary analysis showed that all eight segments of these four viruses are avian-origin and phylogenetically close to the H3N2 canine influenza viruses reported earlier in South Korea. Systematic surveillance is required to monitor the disease and evolutionary behavior of this virus in canine populations in China.

  3. 两种不同病毒载体携带靶向大鼠金属蛋白酶组织抑制因子(TIMP)-1小干扰RNA抗肝纤维化作用的比较%Comparison between the antifibrotic effects of adeno-associated virus and lentivirus carrying small interfering RNA of TIMP-1 in rat liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    马雪梅; 张群; 庞国进; 丛敏

    2013-01-01

    Objective To construct recombinant adeno-associated virus and lentivirus carrying siRNA of TIMP-1 and to investigate their antifibrotic effects on CCl4-induced liver fibrosis in rats.Methods One pair of siRNA which could effectively inhibit expression of the TIMP-1 gene in HSC-T6 was screened and cloned into AAV vector and lentiviral vector to construct the recombinant AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1.AAV/EGFP and Lenti/EGFP as negative control were also obtained.Fifty-eight male Wistar rats were randomly divided into six groups:control group (n =8),CCl4 group,AAV/EGFP,Lenti/EGFP,AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups (all n =10).After the administration of CCl4 for four weeks,liver samples were collected for the immunohistochemical staining and detection of TIMP-1 expression.Results Livers from the control rats showed normal lobular structure around vessels (HE and Masson staining).In contrast,livers from the model,AAV/EGFP and Lenti/EGFP groups showed severe fibrosis,including septal fibrosis,extensive bridging,and fatty degeneration.The expressions of TIMP-1 mRNA and protein were also elevated in the livers from these groups.Compared with the fibrosis model group,the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups showed good preservation of liver lobular architecture and only mild bridging fibrosis,accompanied by decreased expression of TIMP-1 mRNA and protein.Semi-quantitative analysis of the fibrosis stage indicated that most rats in the model,AAV/EGFP and Lenti/EGFP groups were of S3 and S4 (80%),while 20% of the rats were of S5.In contrast,most rats (90%) in the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups were of stages S2 and S3,with only one rat of S4.There was no significant difference between these recombinant virus therapy groups.Conclusions Both AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 can suppress the expression of TIMP-1 in rat fibrotic liver,playing an effective antifibrotic role in the rat liver.%目的 观察以腺相关病

  4. In ovo and in vitro susceptibility of American alligators (Alligator mississippiensis) to avian influenza virus infection.

    Science.gov (United States)

    Temple, Bradley L; Finger, John W; Jones, Cheryl A; Gabbard, Jon D; Jelesijevic, Tomislav; Uhl, Elizabeth W; Hogan, Robert J; Glenn, Travis C; Tompkins, S Mark

    2015-01-01

    Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection. PMID:25380354

  5. Genome Wide Host Gene Expression Analysis in Chicken Lungs Infected with Avian Influenza Viruses

    Science.gov (United States)

    Gandhale, Pradeep N.; Kumar, Himanshu; Kulkarni, Diwakar D.

    2016-01-01

    The molecular pathogenesis of avian influenza infection varies greatly with individual bird species and virus strain. The molecular pathogenesis of the highly pathogenic avian influenza virus (HPAIV) or the low pathogenic avian influenza virus (LPAIV) infection in avian species remains poorly understood. Thus, global immune response of chickens infected with HPAI H5N1 (A/duck/India/02CA10/2011) and LPAI H9N2 (A/duck/India/249800/2010) viruses was studied using microarray to identify crucial host genetic components responsive to these infection. HPAI H5N1 virus induced excessive expression of type I IFNs (IFNA and IFNG), cytokines (IL1B, IL18, IL22, IL13, and IL12B), chemokines (CCL4, CCL19, CCL10, and CX3CL1) and IFN stimulated genes (OASL, MX1, RSAD2, IFITM5, IFIT5, GBP 1, and EIF2AK) in lung tissues. This dysregulation of host innate immune genes may be the critical determinant of the severity and the outcome of the influenza infection in chickens. In contrast, the expression levels of most of these genes was not induced in the lungs of LPAI H9N2 virus infected chickens. This study indicated the relationship between host immune genes and their roles in pathogenesis of HPAIV infection in chickens. PMID:27071061

  6. Spontaenous Avian Leukosis Virus-like lymphomas in specific-pathogen-free chickens inoculated with serotype 2 Marek’s disease virus

    Science.gov (United States)

    Chickens of Avian Disease and Oncology Laboratory (ADOL) line alv6, known to develop spontaneous avian leukosis virus (ALV)-like lymphomas at two years of age or older, were inoculated either in-ovo, or at 1 day of age with strain SB-1 of serotype 2 Marek’s disease virus (MDV). Inoculated and uninoc...

  7. Engineering development of avian influenza virus detection system in a patient's body

    International Nuclear Information System (INIS)

    The avian influenza virus detection equipment in a patient's body has been made. Currently, detection of avian influenza virus carried out by expensive laboratory equipment's, so only certain hospitals can perform this detection. This developing equipment is expected to be cheaper than existing equipment and the diagnosis can be known immediately. The sensing device is made using the principle of nuclear radiation detection. Radiation comes from a drunk labelled tamiflu (oseltamivir) which is drunk to the patient. Tamiflu is a drug to catch, H5N1 viruses in a patient's body. A labelled tamiflu is tamiflu which is labelled by I-131 radioisotopes. The presence of virus in the body is proportional to the amount of radiation captured by the detector. The equipment is composed of a Geiger-Mueller (GM) pancake detector type, a signal processor, a counter, and a data processor (computer). The GM detector converts the radiation that comes into electrical signals. Electrical signal is then converted into TTL level pulses by the signal processor. Pulse counting results are processed by data processor. The total count is proportional to the amount of virus captured by labelled tamiflu. The measurement threshold can be set by medical officer through software. At a certain threshold can be inferred identified patients infected with avian influenza virus. If the measurement below the threshold means that the patient is still within safe limits. This equipment is expected to create avian influenza virus detection system that cheaply and quickly so that more and more hospitals are using to detect the avian influenza virus. (author)

  8. Packaging cells for avian leukosis virus-based vectors with various host ranges.

    OpenAIRE

    Cosset, F L; Ronfort, C.; Molina, R. M.; Flamant, F.; Drynda, A; Benchaibi, M; Valsesia, S; Nigon, V M; Verdier, G

    1992-01-01

    Using our previously described Haydée semipackaging cell line (F. L. Cosset, C. Legras, Y. Chebloune, P. Savatier, P. Thoraval, J. L. Thomas, J. Samarut, V. M. Nigon, and G. Verdier, J. Virol. 64:1070-1078, 1990) which produces avian leukosis virus gag and pol proteins, we have constructed packaging cells with subgroups B, C, and E envelope specificities. This allows us to produce helper-free avian leukosis virus particles carrying the lacZ reporter gene and the A, B, C, or E subgroup specifi...

  9. 9型重组腺相关病毒介导抗核转录因子-κB核酶基因体外转染大鼠心肌细胞及对核转录因子-κB活性的影响%Transfection of rats H9C2 cells with recombinant adeno-associated virus Serotype 9 mediated AntiNF-κB ribozyme in vitro and effects on nuclear factor-κB activity

    Institute of Scientific and Technical Information of China (English)

    高霞; 马依彤; 杨毅宁; 向阳; 陈邦党; 刘芬

    2010-01-01

    Objective To evaluate the transfection efficiency using recombinant adeno-associated virus serotype 9 (rAAV9) mediated anti-nuclear factor-κB (NF) -κB ribozyme and enhanced green fluorescent protein (rAAV9-EGFP-R65) to rats H9C2 cells and the effect on NF-κB activity. Methods rAAV9EGFP-R65 was transfected into H9C2 ceils at multiplicities of infection ( MOI = 1 x 106 v. g./cell). EGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of EGFP positive cells was determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. H9C2 ceils were treated with tumor necrosis factor (TNF)-α, rAAV9-EGFP-R65 and PDTC. The DNA binding activity of NF-KF-κB was examined by electrophoretic mobility shift assay (EMSA). Results The cells began to exhibit EGFP expression one day after transfection. The fluorescence intensity was increased with the time of transfection. EGFP expression reached the maximum on the day 5, at the point of which the transduction efficiency was (32.27 + 3.19)%. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. TNF-α could activate NF-κB, and rAAV9-EGFP-R65 and PDCT could efficiently decrease NF-κB activation in rats H9C2 cells. Conclusion rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition, rAAVg-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro.%目的 观察9型重组腺相关病毒(rAAV9)介导抗核转录因子-κB(NF-κB)核酶基因(rAAV9-ECFP-R65)对大鼠心肌H9C2细胞的转染及对NF-κB活性的影响.方法 rAAV9-EGFP-R65按转染复数(MOI)1×106v.g./cell转染H9C2细胞,在倒置荧光显微镜下观察增强型绿色荧光蛋白(EGFP)阳性表达,采用流式细胞仪检测转染效率.Alamar Blue法检测rAAV9-EGFP-R65对H9C2细胞增殖影响.肿瘤坏死因子-α(TNF-α)、rAAV9

  10. Adeno-Associated Viral Vector-Induced Overexpression of Neuropeptide Y Y2 Receptors in the Hippocampus Suppresses Seizures

    Science.gov (United States)

    Woldbye, David P. D.; Angehagen, Mikael; Gotzsche, Casper R.; Elbrond-Bek, Heidi; Sorensen, Andreas T.; Christiansen, Soren H.; Olesen, Mikkel V.; Nikitidou, Litsa; Hansen, Thomas v. O.; Kanter-Schlifke, Irene; Kokaia, Merab

    2010-01-01

    Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is…

  11. The hemagglutinin structure of an avian H1N1 influenza A virus

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Tianwei; Wang, Gengyan; Li, Anzhang; Zhang, Qian; Wu, Caiming; Zhang, Rongfu; Cai, Qixu; Song, Wenjun; Yuen, Kwok-Yung; (U. Hong Kong); (Inter. Inst. Infect. Imm.); (Xiamen)

    2009-09-15

    The interaction between hemagglutinin (HA) and receptors is a kernel in the study of evolution and host adaptation of H1N1 influenza A viruses. The notion that the avian HA is associated with preferential specificity for receptors with Sia{alpha}2,3Gal glycosidic linkage over those with Sia{alpha}2,6Gal linkage is not all consistent with the available data on H1N1 viruses. By x-ray crystallography, the HA structure of an avian H1N1 influenza A virus, as well as its complexes with the receptor analogs, was determined. The structures revealed no preferential binding of avian receptor analogs over that of the human analog, suggesting that the HA/receptor binding might not be as stringent as is commonly believed in determining the host receptor preference for some subtypes of influenza viruses, such as the H1N1 viruses. The structure also showed difference in glycosylation despite the preservation of related sequences, which may partly contribute to the difference between structures of human and avian origin.

  12. Evaluation of a commercial bELISA serologic assay for avian influenza virus detection in wild birds

    Science.gov (United States)

    Avian influenza (AI) virus surveillance in wild birds is predominately dependent on diagnostic assays that identify the virus, including reverse transcriptase polymerase chain reaction and virus isolation. A sensitive and specific assay to detect AI virus antibodies would complement existing survei...

  13. Broadly protective adenovirus-based multivalent vaccines against highly pathogenic avian influenza viruses for pandemic preparedness.

    Science.gov (United States)

    Vemula, Sai V; Ahi, Yadvinder S; Swaim, Anne-Marie; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K

    2013-01-01

    Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced. PMID:23638099

  14. Landscape attributes driving avian influenza virus circulation in the Lake Alaotra region of Madagascar

    Directory of Open Access Journals (Sweden)

    Laure Guerrini

    2014-05-01

    Full Text Available While the spatial pattern of the highly pathogenic avian influenza H5N1 virus has been studied throughout Southeast Asia, little is known on the spatial risk factors for avian influenza in Africa. In the present paper, we combined serological data from poultry and remotely sensed environmental factors in the Lake Alaotra region of Madagascar to explore for any association between avian influenza and landscape variables. Serological data from cross-sectional surveys carried out on poultry in 2008 and 2009 were examined together with a Landsat 7 satellite image analysed using supervised classification. The dominant landscape features in a 1-km buffer around farmhouses and distance to the closest water body were extracted. A total of 1,038 individual bird blood samples emanating from 241 flocks were analysed, and the association between avian influenza seroprevalence and these landcape variables was quantified using logistic regression models. No evidence of the presence of H5 or H7 avian influenza subtypes was found, suggesting that only low pathogenic avian influenza (LPAI circulated. Three predominant land cover classes were identified around the poultry farms: grassland savannah, rice paddy fields and wetlands. A significant negative relationship was found between LPAI seroprevalence and distance to the closest body of water. We also found that LPAI seroprevalence was higher in farms characterised by predominant wetlands or rice landscapes than in those surrounded by dry savannah. Results from this study suggest that if highly pathogenic avian influenza H5N1 virus were introduced in Madagascar, the environmental conditions that prevail in Lake Alaotra region may allow the virus to spread and persist.

  15. In vivo evaluation of recombinant Vaccinia virus MVA delivering ancestral H9 hemagglutinin antigen of Avian Influenza virus

    OpenAIRE

    Becker, Jens Michael

    2015-01-01

    Avian Influenza (AI) viruses pose a threat to human and animal health and are responsible for potential economic losses. From the waterfowl reservoir, these RNA viruses can be transmitted to domestic poultry and humans, causing illness and death among people as well as mass culling of farm birds worldwide. This study contributes to increasing the knowledge by evaluating a promising poxvirus-based vector vaccine that carries and expresses an artificial, computationally derived hemagglutini...

  16. Inactivation of low pathogenicity notifiable avian influenza virus and lentogenic Newcastle disease virus following pasteurization in liquid egg products

    Science.gov (United States)

    Sixty seven million cases of shell eggs produced per year in the U.S. are processed as liquid egg product. The U.S. also exports a large amount of egg products. Although the U.S. is normally free of avian influenza, concern about contamination of egg product with these viruses has in the past result...

  17. Development of an endogenous virus-free line of chickens susceptible to all subgroups of avian leukosis virus

    Science.gov (United States)

    Primary chicken embryo fibroblasts (CEF) from special specific pathogen free chicken lines are normally used for detection of contamination with avian leukosis viruses (ALV). The suitability and efficiency of such tests mostly depend on the susceptibility of CEF to varied subgroups of ALV. The ideal...

  18. Homo- and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa.

    Directory of Open Access Journals (Sweden)

    Taiana P Costa

    Full Text Available Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin and heterosubtypic (heterologous hemagglutinin low pathogenic avian influenza (LPAI viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.

  19. Detection by PCR of Multiple Subgroups of Avian Leukosis Virus (ALV) in Broilers in the Sudan

    OpenAIRE

    Abdelmelik Ibrahim Khalafalla; Maaz Majzoub Abdel-Latif

    2005-01-01

    An investigation on avian leukosis virus infection in broiler parent farm in Khartoum state, Sudan was conducted. Clinical signs, morbidity rate, mortality rate were recorded. Necropsy was performed, histopathological sections from infected livers were made, and virus isolation trials in chick embryo fibroblast and chorioallantoic membrane were performed. PCR tests were performed on DNA extracted from infected livers and spleen. Affected birds showed in-appetence, abnormal feathering, palenes...

  20. Molecular Characterization of Subtype H11N9 Avian Influenza Virus Isolated from Shorebirds in Brazil

    OpenAIRE

    Hurtado, Renata; Fabrizio, Thomas; Vanstreels, Ralph Eric Thijl; Krauss, Scott; Webby, Richard J.; Webster, Robert G.; Durigon, Edison Luiz

    2015-01-01

    Migratory aquatic birds play an important role in the maintenance and spread of avian influenza viruses (AIV). Many species of aquatic migratory birds tend to use similar migration routes, also known as flyways, which serve as important circuits for the dissemination of AIV. In recent years there has been extensive surveillance of the virus in aquatic birds in the Northern Hemisphere; however in contrast only a few studies have been attempted to detect AIV in wild birds in South America. Ther...

  1. Isolation of avian influenza virus (H9N2) from emu in China

    OpenAIRE

    Kang Wenhua; Pang Wanyong; Hao Junfeng; Zhao Deming

    2006-01-01

    Abstract This is the first reported isolation of avian influenza virus (AIV) from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic ...

  2. Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts.

    OpenAIRE

    Decker, S.

    1981-01-01

    Protein phosphorylation was examined in whole cell extracts from normal and avian sarcoma virus-transformed chicken embryo fibroblasts. The addition of serum or epidermal growth factor to serum-starved normal cells resulted in increased 32P labeling of a Mr 30,000 protein. In extracts from cells transformed by a temperature-sensitive mutant of Schmidt-Ruppin virus, subgroup A, and grown at the permissive temperature, the protein was phosphorylated regardless of serum starvation. This Mr 30,00...

  3. Two special topics on the avian influenza virus and on epigenetics,have drawn much attention

    Institute of Scientific and Technical Information of China (English)

    HU YongLin

    2010-01-01

    @@ Several excellent well-organized reviews and research papers on two special topics, "The challenges of avian influenza virus: mechanism, epidemiology, and control" and "Molecular epigenetics: dawn of a new era of biomedical research", published in the 2009 edition of Science in China Series C: Life Sciences, have drawn much attention.

  4. Avian leukosis virus type J (ALV-J) in the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Jurajda, V.; Kulíková, L.; Halouzka, R.; Geryk, Josef; Svoboda, Jan

    2000-01-01

    Roč. 69, č. 2 (2000), s. 143-145. ISSN 0001-7213 R&D Projects: GA ČR GA524/01/0866 Keywords : avian leukosis virus-J * myelocytomatosis * breeding chickens Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.240, year: 2000

  5. Aberrant expression of liver microRNA in chickens infected with subgroup J avian leukosis virus

    Science.gov (United States)

    Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus primarily causing myeloid leukosis (ML) in broilers. Although ALV is well under control in a few countries including the U.S.A., poultry industry in many parts of the world continues suffering from serious economic loss due to sporad...

  6. Diversity in Avian Leukosis Virus Isolates From Single Outbreak of Myelocytomatosis in Commercial Layers

    Science.gov (United States)

    In 1997, three white leghorn flocks were diagnosed with the first reported case of myelocytomatosis in commercial and field layer flocks. Moreover, the first naturally occurring recombinant avian leukosis virus (ALV) termed AF 115-4 (ALV-B/J) containing the envelope of ALV-B and long terminal repeat...

  7. EXPRESSION EFFECT OF RECOMBINANT ENVELOPE GENE OF AVIAN LEUKOSIS VIRUS SUBGROUP J IN SF 9 CELLS

    Science.gov (United States)

    Expression effect of envelope gene of avian leukosis virus (ALV-J) in Sf9 cells infected with recombinant baculovirus rBac-env was analyzed by immunofluorescent assay and immunoprecipitation. The results showed that recombinant envelope gene product was a glycosylated protein in tunicumycin treatme...

  8. Mekabu fucoidan: Structural complexity and defensive effects against avian influenza A viruses

    Czech Academy of Sciences Publication Activity Database

    Synytsya, A.; Bleha, R.; Synytsya, Al.; Pohl, Radek; Hayashi, K.; Yoshinaga, K.; Nakano, T.; Hayashi, T.

    2014-01-01

    Roč. 111, Oct 13 (2014), s. 633-644. ISSN 0144-8617 Institutional support: RVO:61388963 Keywords : Mekabu fucoidan * sporophyll Undaria pinnatifida * spectroscopic methods * avian influenza A virus * immunostimulating effect Subject RIV: CC - Organic Chemistry Impact factor: 4.074, year: 2014

  9. Avian influenza virus with Hemagglutinin-Neuraminidase combination H8N8, isolated in Russia

    Science.gov (United States)

    This study reports the genome sequence of an avian influenza virus (AIV) subtype H8N8 isolated in Russia. The genome analysis shows that all genes belong to AIV Eurasian lineages. The PB2 gene was similar to a Mongolian low pathogenic (LP) AIV H7N1 and a Chinese high pathogenic (HP) AIV H5N2....

  10. Surveillance for avian influenza viruses in wild birds in Denmark and Greenland

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Breum, Solvej Østergaard; Trebbien, Ramona; Handberg, Kurt J.; Therkildsen, Ole R.; Madsen, Jesper J.; Thorup, Kasper; Baroch, John A.; DeLiberto, Thomas J.; Larsen, Lars Erik; Jørgensen, Poul Henrik

    Avian influenza (AI) is a disease of major threat to poultry production. Surveillance of AI in wild birds contributes to the control of AI. In Denmark (DK) and Greenland (GL), extensive surveillance of AI viruses in the wild bird population has been conducted. The surveillance aimed at detecting...

  11. Detection of American lineage low pathogenic avian influenza viruses in Uria lomvia in Greenland

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Hartby, Christina Marie; Krog, Jesper Schak; Holm, Elisabeth; Larsen, Lars Erik

    Denmark. Five birds were randomly selected for diagnostic investigation and samples were taken from the cadavers (pooled oropharyngeal swabs, cloacal swabs, lung/trachea/heart tissues and liver/spleen/kidney tissues, and separate preparation of stomach from a single bird). Avian influenza virus (AIV) with...

  12. Complete Genome Sequence of a New H9N2 Avian Influenza Virus Isolated in China

    OpenAIRE

    Wang, Jing-Yu; Ren, Juan-Juan; Liu, Wan-Hua; Tang, Pan; Wu, Ning; Wang, Chi-Young; Chang, Ching-Dong; Liu, Hung-Jen

    2013-01-01

    The complete genomic sequence of a new H9N2 avian influenza virus (AIV), isolated in northwestern China, was determined. Sequence and phylogenetic analyses based on the sequences of eight genomic segments revealed that the isolate is phylogenetically related to the Y280-like sublineage.

  13. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus

    Science.gov (United States)

    Vaccination against avian influenza (AI) virus, a powerful tool for control of the disease, may result in issues related to surveillance programs and international trade of poultry and poultry products. The use of AI vaccination in poultry would have greater world-wide acceptance if a reliable test...

  14. Fowl plague virus replication in mammalian cell-avian erythrocyte heterokaryons: studies concerning the actinomycin D and ultra-violet lig sensitive phase in influenza virus replication

    International Nuclear Information System (INIS)

    The replication of fowl plague virus in BHK and L cells specifically blocked prior to infection with inhibitors of influenza virus replication (actinomycin D and ultraviolet light irradiation) has been studied by the introduction of a metabolically dormant avian erythrocyte nucleus. This permits the synthesis of just the influenza virus nucleoprotein in actinomycin D (but not ultraviolet light) blocked cells. The NP antigen is first detected in the avian erythrocyte nucleus and subsequently in the heterokaryon cytoplasm

  15. Development of FPV140 antigen-specific ELISA differentiating fowlpox virus isolates from all other viral pathogens of avian origin.

    Science.gov (United States)

    Li, G; Hong, Q; Ren, Y; Lillehoj, H S; He, C; Ren, X

    2012-10-01

    The FPV140 gene encodes an envelope protein of fowlpox virus (FPV). In this study, the FPV140 gene of FPV Chinese isolate HH2008 was cloned and the comparison of its sequence with other FPV isolates showed it to be highly conserved across all FPV isolates. A recombinant plasmid pET-FPV140 carrying FPV140 gene was constructed and transformed into Escherichia coli. The optimal expression condition for the FPV140 gene was developed and purified FPV140 recombinant protein was used to produce rabbit polyclonal antibody. An indirect ELISA using this anti-FPV140 polyclonal antibody was capable of distinguishing avian FPV isolates from other common avian pathogens such as mycoplasma gallisepticum, infectious laryngotracheitis virus, avian influenza virus, infectious bursal disease virus, and avian infectious bronchitis virus. This ELISA will serve as a useful diagnostic tool for the detection of FPV in clinical samples. PMID:22991535

  16. Serological Evidence of Inter-Species Transmission of H9N2 Avian Influenza Virus in Poultry, Iran

    Directory of Open Access Journals (Sweden)

    Mohammad Mehdi Hadipour

    2011-02-01

    Full Text Available Ducks and in-contact backyard chickens on 20 smallholder backyard farms in 4 districts of Shiraz, Southwest of Iran, were monitored for antibodies against H9N2 avian influenza virus using hemagglutinationinhibition (HI test. A total of 200 unvaccinated ducks and backyard chickens were sampled. The mean H I titers and seroprevalence in ducks and backyard chickens were 8.3, 5.7 and 78.4, 62.9%, respectively. Results of this study revealed that the Scavenging ducks are the natural reservoir of avian influenza viruses and play an important role in the epidemiology of H9N2 avian influenza virus infection.

  17. The passage of cells can improve the detection rate of avian leukosis virus to facilitate the elimination of avian leukosis in chickens

    OpenAIRE

    Wang, Xiuzhen; Wang, Bo; Zhang, Peipei; Cheng, Hegang; Sun, Shuhong

    2013-01-01

    Avian leukosis (AL) is one of the most harmful diseases to the poultry industry in China. The detection of the avian leukosis virus (ALV) p27 antigen plays a decisive role in the elimination of avian leukosis. To explore the influence of passaging cells on the detection rate of the ALV p27 antigen, 21 aseptic anticoagulated blood samples were collected from 21 chickens for which the cloacal swabs were positive for the p27 antigen to inoculate two sets of cell culture plates containing DF1 cel...

  18. A Review of the Antiviral Susceptibility of Human and Avian Influenza Viruses over the Last Decade

    Directory of Open Access Journals (Sweden)

    Ding Yuan Oh

    2014-01-01

    Full Text Available Antivirals play an important role in the prevention and treatment of influenza infections, particularly in high-risk or severely ill patients. Two classes of influenza antivirals have been available in many countries over the last decade (2004–2013, the adamantanes and the neuraminidase inhibitors (NAIs. During this period, widespread adamantane resistance has developed in circulating influenza viruses rendering these drugs useless, resulting in the reliance on the most widely available NAI, oseltamivir. However, the emergence of oseltamivir-resistant seasonal A(H1N1 viruses in 2008 demonstrated that NAI-resistant viruses could also emerge and spread globally in a similar manner to that seen for adamantane-resistant viruses. Previously, it was believed that NAI-resistant viruses had compromised replication and/or transmission. Fortunately, in 2013, the majority of circulating human influenza viruses remain sensitive to all of the NAIs, but significant work by our laboratory and others is now underway to understand what enables NAI-resistant viruses to retain the capacity to replicate and transmit. In this review, we describe how the susceptibility of circulating human and avian influenza viruses has changed over the last ten years and describe some research studies that aim to understand how NAI-resistant human and avian influenza viruses may emerge in the future.

  19. Cocirculation of avian H9N2 and contemporary "human" H3N2 influenza A viruses in pigs in southeastern China: Potential for genetic reassortment?

    OpenAIRE

    Peiris, J. S. M.; Guan, Y.; Markwell, D; Ghose, P.; Webster, R G; Shortridge, K. F.

    2001-01-01

    Pigs are permissive to both human and avian influenza viruses and have been proposed to be an intermediate host for the genesis of pandemic influenza viruses through reassortment or adaptation of avian viruses. Prospective virological surveillance carried out between March 1998 and June 2000 in Hong Kong, Special Administrative Region, People's Republic of China, on pigs imported from southeastern China, provides the first evidence of interspecies transmission of avian H9N2 viruses to pigs an...

  20. Amantadine resistance among highly pathogenic avian influenza viruses (H5N1) isolated from India.

    Science.gov (United States)

    Jacob, Aron; Sood, Richa; Chanu, Kh Victoria; Bhatia, Sandeep; Khandia, Rekha; Pateriya, A K; Nagarajan, S; Dimri, U; Kulkarni, D D

    2016-02-01

    Emergence of antiviral resistance among H5N1 avian influenza viruses is the major challenge in the control of pandemic influenza. Matrix 2 (M2) inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir) are the two classes of antiviral agents that are specifically active against influenza viruses and are used for both treatment and prophylaxis of influenza infections. Amantadine targets the M2 ion channel of influenza A virus and interrupts virus life cycle through blockade of hydrogen ion influx. This prevents uncoating of the virus in infected host cells which impedes the release of ribonucleoprotein required for transcription and replication of virion in the nucleus. The present study was carried out to review the status of amantadine resistance in H5N1 viruses isolated from India and to study their replicative capability. Results of the study revealed resistance to amantadine in antiviral assay among four H5N1 viruses out of which two viruses had Serine 31 Asparagine (AGT-AAT i.e., S31N) mutation and two had Valine 27 Alanine (GTT-GCT i.e., V27A) mutation. The four resistant viruses not only exhibited significant difference in effective concentration 50% (EC50) values of amantadine hydrochloride from that of susceptible viruses (P < 0.0001) but also showed significant difference between two different types (S31N and V27A) of mutant viruses (P < 0.05). Resistance to amantadine could also be demonstrated in a simple HA test after replication of the viruses in MDCK cells in presence of amantadine. The study identifies the correlation between in vitro antiviral assay and presence of established molecular markers of resistance, the retention of replicative capacity in the presence of amantadine hydrochloride by the resistant viruses and the emergence of resistant mutations against amantadine among avian influenza viruses (H5N1) without selective drug pressure. PMID:26639679

  1. Pathogenicity, Transmission and Antigenic Variation of H5N1 Highly Pathogenic Avian Influenza Viruses

    Science.gov (United States)

    Jiao, Peirong; Song, Hui; Liu, Xiaoke; Song, Yafen; Cui, Jin; Wu, Siyu; Ye, Jiaqi; Qu, Nanan; Zhang, Tiemin; Liao, Ming

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) was one of the most important avian diseases in poultry production of China, especially in Guangdong province. In recent years, new H5N1 highly pathogenic avian influenza viruses (HPAIV) still emerged constantly, although all poultry in China were immunized with H5N1 vaccinations compulsorily. To better understand the pathogenicity and transmission of dominant clades of the H5N1 HPAIVs in chicken from Guangdong in 2012, we chose a clade 7.2 avian influenza virus named A/Chicken/China/G2/2012(H5N1) (G2) and a clade 2.3.2.1 avian influenza virus named A/Duck/China/G3/2012(H5N1) (G3) in our study. Our results showed that the chickens inoculated with 103 EID50 of G2 or G3 viruses all died, and the titers of virus replication detected in several visceral organs were high but different. In the naive contact groups, virus shedding was not detected in G2 group and all chickens survived, but virus shedding was detected in G3 group and all chickens died. These results showed that the two clades of H5N1 HPAIVs had high pathogenicity in chickens and the contact transmission of them was different in chickens. The results of cross reactive HI assay showed that antigens of G2 and G3 were very different from those of current commercial vaccines isolates (Re-4, Re-6, and D7). And to evaluate the protective efficacy of three vaccines against most isolates form Guangdong belonging to clade 2.3.2.1 in 2012, G3 was chosen to challenge the three vaccines such as Re-4, Re-6, and D7. First, chickens were immunized with 0.3 ml Re-4, Re-6, and D7 inactivated vaccines by intramuscular injection, respectively, and then challenged with 106 EID50 of G3 on day 28 post-vaccination. The D7 vaccine had 100% protection against G3 for chickens, the Re-6 vaccine had 88.9%, and the Re-4 vaccine only had 66.7%. Our results suggested that the D7 vaccine could prevent and control H5N1 virus outbreaks more effectively in Guangdong. From the above, it was

  2. Evolution of highly pathogenic avian H5N1 influenza viruses

    Energy Technology Data Exchange (ETDEWEB)

    Macken, Catherine A [Los Alamos National Laboratory; Green, Margaret A [Los Alamos National Laboratory

    2009-01-01

    Highly pathogenic avian H5N1 viruses have circulated in Southeast Asia for more than a decade, are now endemic in parts of this region, and have also spread to more than 60 countries on three continents. The evolution of these viruses is characterized by frequent reassortment events that have created a significant number of different genotypes, both transient and longer lasting. However, fundamental questions remain about the generation and perpetuation of this substantial genetic diversity. These gaps in understanding may, in part, be due to the difficulties of genotyping closely related viruses, and limitations in the size of the data sets used in analysis. Using our recently published novel genotyping procedure ('two-time test'), which is amenable to high throughput analysis and provides an increased level of resolution relative to previous analyses, we propose a detailed model for the evolution and diversification of avian H5N1 viruses. Our analysis suggests that (i) all current H5N1 genotypes are derived from a single, clearly defined sequence of initial reassortment events; (ii) reassortment of the polymerase and NP genes may have played an important role in avian H5N1 virus evolution; (iii) the current genotype Z viruses have diverged into three distinguishable sub-genotypes in the absence of reassortment; (iv) some potentially significant molecular changes appear to be correlated with particular genotypes (for example, reassortment of the internal genes is often paralleled by a change in the HA clade); and (v) as noted in earlier studies of avian influenza A virus evolution, novel segments are typically derived from different donors (i.e., there is no obvious pattern of gene linkage in reassortment). The model of avian H5N1 viral evolution by reassortment and mutation that emerges from our study provides a context within which significant amino acid changes may be revealed; it also may help in predicting the 'success' of newly emerging

  3. Little evidence of subclinical avian influenza virus infections among rural villagers in Cambodia.

    Directory of Open Access Journals (Sweden)

    Gregory C Gray

    Full Text Available In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI. Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI virus infection and withdrew from the study. Ninety-seven ILI cases (22.1% were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0% had detectable antibody titers (≥ 1:10 against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6, 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1, 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up against an avian-like A/Hong Kong/1073/1999(H9N2, 6 (1 detected at both 12- and 24-month follow-up against an avian-like A/Duck/Memphis/546/74(H11N9, and 2 against an avian-like A/Duck/Alberta/60/76(H12N5. With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these

  4. Complete Genome Sequences of Six Avian-Like H1N1 Swine Influenza Viruses from Northwestern China

    OpenAIRE

    Wang, Jing-Yu; Ren, Juan-Juan; Qiu, Yuan-Hao; Liu, Hung-Jen

    2013-01-01

    Very little is known about swine influenza in northwestern China. Here, we report the complete genomic sequences of six avian-like H1N1 swine influenza viruses (SIVs) isolated in pigs in northwestern China. Phylogenetic analyses of the sequences of eight genomic segments demonstrated that they are avian-like H1N1 SIVs.

  5. The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element

    DEFF Research Database (Denmark)

    Bakhshesh, M.; Groppelli, E.; Willcocks, M.A.; Royall, E.; Belsham, Graham; Roberts, L.O.

    2008-01-01

    Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide...... also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in...

  6. The Development of Pathogenicity of Avian Influenza Virus Isolated from Indonesia

    Directory of Open Access Journals (Sweden)

    Michael Haryadi Wibowo

    2015-11-01

    Full Text Available Highly pathogenic avian infl uenza outbreak in Indonesia has been reported in various poultry due toH5N1 subtype. The presence of multiple basic amino acids within the cleavage site of HA glycoprotein hasbeen identifi ed to be associated with the pathogenicity of avian infl uenza virus. The study was retrospectivestudy which was designed to characterize the cleavage site and fusion site region of haemagglutinin gene ofAIV isolated from various poultry in 2003 to 2013. Isolation, Identifi cation and propagation were carried outto collect viral stock. For virus detection, reverse transcriptase PCR (RT-PCR method on H5 and N1 genefragment was performed. All of RT-PCR HA gene positive products were sequenced for further nucleotideanalysis and to determine the nucleotide composition at the targeted fragment. The results are all AIV isolateswere identifi ed as H5N1 subtype. The sequence analyses revealed some motives of basic amino acid motivethat were classifi ed as highly pathogenic avian infl uenza virus. Further analyses on fusion domain of all AIVisolated during the period 2003 to 2013 showed conserved amino acid.Keywords: avian infl uenza, haemagglutinin, cleavage site, basic amino acid, fusion site

  7. Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus

    OpenAIRE

    Chai, Ning; Bates, Paul

    2006-01-01

    Subgroup J avian leukosis virus (ALV-J) is a recently identified avian oncogenic retrovirus responsible for severe economic losses worldwide. In contrast with the other ALV subgroups, ALV-J predominantly induces myeloid leukosis in meat-type chickens. Despite significant homology with the other ALV subgroups across most of the genome, the envelope protein of ALV-J (EnvJ) shares low homology with the others. Pathogenicity and myeloid leukosis induction map to the env gene of ALV-J. A chimeric ...

  8. Quantification and characterization of avian RNA tumor virus group specific antigen by radioimmunoassay

    International Nuclear Information System (INIS)

    Although the gs antigen preparation used in the RIA had originally been thought to contain only one protein, further analysis by column chromatography and polyacrylamide gel electrophoresis revealed the presence of two viral polypeptides. Subsequent analysis of the immune precipitates of the RIA by SDS polyacrylamide gel electrophoresis identified 70 percent of precipitable radioactivity as gs-1 (major antigen) and 30 percent as gs-3. Extensive immunological competitive inhibition reactions confirmed this analysis. The RIA for avian group specific antigen has, therefore, duel specificity since it does not require one polypeptide specificity for the majority of its applications. The RIA was used to follow the progress of in vitro infection by avian leukosis and sarcoma viruses by monitoring the intracellular appearance of gs antigen. In these studies gs antigen was detected within 6-12 hours after virus infections. This was 6 hours before the release of progeny virus and 18 hours before the previous studies had detected viral products. These studies were also important in demonstrating the feasibility of using production of an intracellular viral antigen as a marker to follow virus infections. The success of these in vitro studies allowed the quantification in vivo of the pathogenesis of a virus induced chicken leukemia, avian myeloblastosis

  9. The infection of chicken tracheal epithelial cells with a H6N1 avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Ching-I Shen

    Full Text Available Sialic acids (SAs linked to galactose (Gal in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1-3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry.

  10. Interspecies transmission and host restriction of avian H5N1 influenza virus

    Institute of Scientific and Technical Information of China (English)

    GAO; George; Fu

    2009-01-01

    Long-term endemicity of avian H5N1 influenza virus in poultry and continuous sporadic human infections in several countries has raised the concern of another potential pandemic influenza. Suspicion of the avian origin of the previous pandemics results in the close investigation of the mechanism of interspecies transmission. Entry and fusion is the first step for the H5N1 influenza virus to get into the host cells affecting the host ranges. Therefore receptor usage study has been a major focus for the last few years. We now know the difference of the sialic acid structures and distributions in different species, even in the different parts of the same host. Many host factors interacting with the influenza virus component proteins have been identified and their role in the host range expansion and interspecies transmission is under detailed scrutiny. Here we review current progress in the receptor usage and host factors.

  11. New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks

    DEFF Research Database (Denmark)

    Bragstad, K.; Jørgensen, Poul Henrik; Handberg, Kurt; Mellergaard, Stig; Corbet, S.; Fomsgaard, A.

    2005-01-01

    7, was identified. The HA gene showed great. sequence similarity to the highly pathogenic avian influenza A virus (HPAIV) A/Chicken/ftaly/312/97 (H5N2); however, the cleavage site sequence between HA1 and HA2 had a motif typical for low pathogenic avian influenza viruses (LPAIV). The full-length NA......During the past years increasing incidences of influenza A zoonosis have made it of uppermost importance to possess methods for rapid and precise identification and characterisation of influenza A Viruses. We present here a convenient one-step RT-PCR method that will amplify full......-length haemagglutinin (HA) and neuraminidase (NA) directly from clinical samples and from all known subtypes of influenza A. We applied the method on samples collected in September 2003 from a Danish flock of mallards with general health problems and by this a previously undescribed influenza A subtype combination, H5N...

  12. Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

    Science.gov (United States)

    Harrington, Elizabeth A; Sloan, Jennifer L; Manoli, Irini; Chandler, Randy J; Schneider, Mark; McGuire, Peter J; Calcedo, Roberto; Wilson, James M; Venditti, Charles P

    2016-05-01

    Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p gene delivery as a treatment for mut MMA. PMID:26790480

  13. Environmental and demographic determinants of avian influenza viruses in waterfowl across the contiguous United States.

    Directory of Open Access Journals (Sweden)

    Matthew L Farnsworth

    Full Text Available Outbreaks of avian influenza in North American poultry have been linked to wild waterfowl. A first step towards understanding where and when avian influenza viruses might emerge from North American waterfowl is to identify environmental and demographic determinants of infection in their populations. Laboratory studies indicate water temperature as one determinant of environmental viral persistence and we explored this hypothesis at the landscape scale. We also hypothesized that the interval apparent prevalence in ducks within a local watershed during the overwintering season would influence infection probabilities during the following breeding season within the same local watershed. Using avian influenza virus surveillance data collected from 19,965 wild waterfowl across the contiguous United States between October 2006 and September 2009 We fit Logistic regression models relating the infection status of individual birds sampled on their breeding grounds to demographic characteristics, temperature, and interval apparent prevalence during the preceding overwintering season at the local watershed scale. We found strong support for sex, age, and species differences in the probability an individual duck tested positive for avian influenza virus. In addition, we found that for every seven days the local minimum temperature fell below zero, the chance an individual would test positive for avian influenza virus increased by 5.9 percent. We also found a twelve percent increase in the chance an individual would test positive during the breeding season for every ten percent increase in the interval apparent prevalence during the prior overwintering season. These results suggest that viral deposition in water and sub-freezing temperatures during the overwintering season may act as determinants of individual level infection risk during the subsequent breeding season. Our findings have implications for future surveillance activities in waterfowl and domestic

  14. Bronchointerstitial pneumonia in guinea pigs following inoculation with H5N1 high pathogenicity avian influenza virus

    Science.gov (United States)

    The H5N1 high pathogenicity avian influenza (HPAI) viruses have caused widespread disease of poultry in Asia, Africa and the Middle East, and sporadic human infections. The guinea pig model has been used to study human H3N2 and H1N1 influenza viruses, but knowledge is lacking on H5N1 HPAI virus inf...

  15. Receptor Characterization and Susceptibility of Cotton Rats to Avian and 2009 Pandemic Influenza Virus Strains

    OpenAIRE

    Blanco, Jorge C. G.; Pletneva, Lioubov M; Wan, Hongquan; Araya, Yonas; Angel, Matthew; Oue, Raymonde O.; Sutton, Troy C.; Perez, Daniel R

    2013-01-01

    Animal influenza viruses (AIVs) are a major threat to human health and the source of pandemic influenza. A reliable small-mammal model to study the pathogenesis of infection and for testing vaccines and therapeutics against multiple strains of influenza virus is highly desirable. We show that cotton rats (Sigmodon hispidus) are susceptible to avian and swine influenza viruses. Cotton rats express α2,3-linked sialic acid (SA) and α2,6-linked SA residues in the trachea and α2,6-linked SA residu...

  16. Filter-feeding bivalves can remove avian influenza viruses from water and reduce infectivity

    OpenAIRE

    Faust, Christina; Stallknecht, David; Swayne, David; Brown, Justin

    2009-01-01

    Avian influenza (AI) viruses are believed to be transmitted within wild aquatic bird populations through an indirect faecal–oral route involving contaminated water. This study examined the influence of filter-feeding bivalves, Corbicula fluminea, on the infectivity of AI virus in water. Clams were placed into individual flasks with distilled water inoculated 1:100 with a low pathogenic (LP) AI virus (A/Mallard/MN/190/99 (H3N8)). Viral titres in water with clams were significantly lower at 24 ...

  17. Genomic sequences of human infection of avian-origin influenza A(H7N9) virus in Zhejiang province

    Institute of Scientific and Technical Information of China (English)

    陈寅

    2013-01-01

    Objective To analyze the etiology and genomic sequences of human infection of avian-origin influenza A (H7N9) virus from Zhejiang province.Methods Viral RNA was extracted from patients of suspected H7N9

  18. H9N2 avian influenza virus-derived natural reassortant H5N2 virus in swan containing the hemagglutinin segment from Eurasian H5 avian influenza virus with an in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site.

    Science.gov (United States)

    Wang, Youling; Yuan, Xiaoyuan; Qi, Lihong; Zhang, Yuxia; Xu, Huaiying; Yang, Jinxing; Ai, Wu; Qi, Wenbao; Liao, Ming; Wang, Dan; Song, Minxun; Li, Feng

    2016-06-01

    We isolated a novel H5N2 avian influenza virus from swans in China. The virus was derived from a widespread H9N2 avian influenza virus but acquired the hemagglutinin gene from Eurasian H5 subtype with a naturally occurring in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site. PMID:26910357

  19. Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products

    OpenAIRE

    Werling, Natalie Jayne; Satkunanathan, Stifani; Thorpe, Robin; Zhao, Yuan

    2015-01-01

    Abstract Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target se...

  20. Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges

    OpenAIRE

    Raj, Deepak; Davidoff, Andrew M.; Nathwani, Amit C.

    2011-01-01

    Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated...

  1. Generation of Insulin-Producing Human Mesenchymal Stem Cells Using Recombinant Adeno-Associated Virus

    OpenAIRE

    Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

    2007-01-01

    The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet β-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced wi...

  2. Regression of Schwannomas Induced by Adeno-Associated Virus-Mediated Delivery of Caspase-1

    OpenAIRE

    Prabhakar, Shilpa; Taherian, Mehran; Gianni, Davide; Conlon, Thomas J.; Fulci, Giulia; Brockmann, Jillian; Stemmer-Rachamimov, Anat; Sena-Esteves, Miguel; Breakefield, Xandra O.; Brenner, Gary J.

    2012-01-01

    Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene...

  3. Restriction Factors Against Recombinant Adeno-associated Virus Vectormediated Gene Transfer in Dystrophin-deficient Muscles.

    Science.gov (United States)

    Dupont, Jean-Baptiste

    2016-01-01

    Despite the unprecedented beneficial effects of rAAV gene therapy in animal models of Duchenne muscular dystrophy (DMD), the need to inject large amounts of vector in vivo to improve phenotype raises obvious biosafety concerns. While rAAV vectors generally exhibit a good safety profile, specific pathological phenotypes such as those observed in dystrophin-deficient muscles may promote immunotoxic/genotoxic effects. Increasing the therapeutic index of rAAV in DMD muscles by reducing the effective dose could be a pivotal means of ensuring efficient clinical translation. This requires a comprehensive understanding of the rAAV transduction process, which is almost always studied in non-pathological tissues or in vitro. In this review, we focus on the molecular fate of rAAV after injection, and how the individual stages of transduction could be affected in the context of DMD. PMID:27121109

  4. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  5. gga-miR-375 Plays a Key Role in Tumorigenesis Post Subgroup J Avian Leukosis Virus Infection

    OpenAIRE

    Li, Hongxin; Shang, Huiqing; Shu, Dingming; Zhang, Huanmin; Ji, Jun; Sun, Baoli; Li, Hongmei; Xie, Qingmei

    2014-01-01

    Avian leukosis is a neoplastic disease caused in part by subgroup J avian leukosis virus J (ALV-J). Micro ribonucleic acids (miRNAs) play pivotal oncogenic and tumour-suppressor roles in tumour development and progression. However, little is known about the potential role of miRNAs in avian leukosis tumours. We have found a novel tumour-suppressor miRNA, gga-miR-375, associated with avian leukosis tumorigenesis by miRNA microarray in a previous report. We have also previously studied the biol...

  6. Transmission of avian influenza A viruses among species in an artificial barnyard.

    Directory of Open Access Journals (Sweden)

    Jenna E Achenbach

    Full Text Available Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks or seroconversion (other species over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.

  7. Molecular analysis of the c-myc locus in normal tissue and in avian leukosis virus-induced lymphomas.

    OpenAIRE

    Neel, B G; Gasic, G P; Rogler, C E; Skalka, A M; Ju, G; Hishinuma, F; Papas, T; Astrin, S M; Hayward, W S

    1982-01-01

    We isolated molecular clones of the provirus-host cell junctions (tumor junction fragments) from two avian leukosis virus-induced lymphomas and compared the structures of these clones with a clone of the normal c-myc gene. Restriction mapping and DNA sequencing demonstrated that normal proviral integration events occurred adjacent to c-myc in both tumors, without gross structural alteration of c-myc. The right long terminal repeat of an avian leukosis virus provirus is integrated upstream fro...

  8. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    International Nuclear Information System (INIS)

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product

  9. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

    OpenAIRE

    Hahn, M; Hayward, W S

    1988-01-01

    We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  10. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  11. Infection with Possible Precursor of Avian Influenza A(H7N9) Virus in a Child, China, 2013

    OpenAIRE

    Ren, Lili; Yu, Xuelian; Zhao, Baihui; Wu, Fan; Jin, Qi; Zhang, Xi; Wang, Jianwei

    2014-01-01

    During the early stage of the avian influenza A(H7N9) epidemic in China in March 2013, a strain of the virus was identified in a 4-year-old boy with mild influenza symptoms. Phylogenetic analysis indicated that this strain, which has similarity to avian subtype H9N2 viruses, may represent a precursor of more-evolved H7N9 subtypes co-circulating among humans.

  12. The pathogenesis of low pathogenicity H7 avian influenza viruses in chickens, ducks and turkeys

    Directory of Open Access Journals (Sweden)

    Pope Conrad R

    2010-11-01

    Full Text Available Abstract Background Avian influenza (AI viruses infect numerous avian species, and low pathogenicity (LP AI viruses of the H7 subtype are typically reported to produce mild or subclinical infections in both wild aquatic birds and domestic poultry. However relatively little work has been done to compare LPAI viruses from different avian species for their ability to cause disease in domestic poultry under the same conditions. In this study twelve H7 LPAI virus isolates from North America were each evaluated for their comparative pathogenesis in chickens, ducks, and turkeys. Results All 12 isolates were able to infect all three species at a dose of 106 50% egg infectious doses based on seroconversion, although not all animals seroconverted with each isolate-species combination. The severity of disease varied among isolate and species combinations, but there was a consistent trend for clinical disease to be most severe in turkeys where all 12 isolates induced disease, and mortality was observed in turkeys exposed to 9 of the 12 viruses. Turkeys also shed virus by the oral and cloacal routes at significantly higher titers than either ducks or chickens at numerous time points. Only 3 isolates induced observable clinical disease in ducks and only 6 isolates induced disease in chickens, which was generally very mild and did not result in mortality. Full genome sequence was completed for all 12 isolates and some isolates did have features consistent with adaptation to poultry (e.g. NA stalk deletions, however none of these features correlated with disease severity. Conclusions The data suggests that turkeys may be more susceptible to clinical disease from the H7 LPAI viruses included in this study than either chickens or ducks. However the severity of disease and degree of virus shed was not clearly correlated with any isolate or group of isolates, but relied on specific species and isolate combinations.

  13. Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human.

    Science.gov (United States)

    Li, Jun; Yu, Xinfen; Pu, Xiaoying; Xie, Li; Sun, Yongxiang; Xiao, Haixia; Wang, Fenjuan; Din, Hua; Wu, Ying; Liu, Di; Zhao, Guoqiu; Liu, Jun; Pan, Jingcao

    2013-06-01

    A novel H7N9 influenza A virus has been discovered as the causative identity of the emerging acute respiratory infection cases in Shanghai, China. This virus has also been identified in cases of infection in the neighboring area Hangzhou City in Zhejiang Province. In this study, epidemiologic, clinical, and virological data from three patients in Hangzhou who were confirmed to be infected by the novel H7N9 influenza A virus were collected and analyzed. Human respiratory specimens and chicken feces from a contacted free market were tested for influenza virus by real-time reverse transcription PCR (RT-PCR) and sequencing. The clinical features of the three cases were similar featured with high fever and severe respiratory symptoms; however, only one of the patients died. A certain degree of diversity was observed among the three Hangzhou viruses sequenced from human samples compared with other reported H7N9 influenza A viruses. The sequences of the novel avian-origin H7N9 influenza viruses from Hangzhou City contained important amino acid substitutions related to human adaptation. One of the Hangzhou viruses had gained a novel amino acid substitution (Q226I) in the receptor binding region of hemagglutinin. More importantly, the virus sequenced from the chicken feces had a 627E substitution in the PB2 protein instead of the mammalian-adapted 627K substitution that was found in the PB2 proteins from the Hangzhou viruses from the three patients. Therefore, the newly-emerging H7N9 virus might be under adaptation pressure that will help it "jump" from avian to human hosts. The significance of these substitutions needs further exploration, with both laboratory experiments and extensive field surveillance. PMID:23657795

  14. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Stoker, A.W.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1988-03-01

    The authors have constructed an avian leukosis virus derivative with a 5{prime} deletion extending from within the tRNA primer binding site to a SacI site in the leader region. The aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1{Psi}{sup {minus}}, to develop vector-packaging cell lines. They show that RAV-1{Psi}{sup {minus}} can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, they have demonstrated that QT6-derived lines expressing RAV-1{Psi}{sup {minus}} can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-{Psi}{sup {minus}}-expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. They show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.

  15. Occurrence of avian Plasmodium and West Nile virus in culex species in Wisconsin

    Science.gov (United States)

    Hughes, T.; Irwin, P.; Hofmeister, E.; Paskewitz, S.M.

    2010-01-01

    The occurrence of multiple pathogens in mosquitoes and birds could affect the dynamics of disease transmission. We collected adult Culex pipiens and Cx. restuans (Cx. pipiens/restuans hereafter) from sites in Wisconsin and tested them for West Nile virus (WNV) and for avian malaria (Plasmodium). Gravid Cx. pipiens/restuans were tested for WNV using a commercial immunoassay, the RAMP?? WNV test, and positive results were verified by reverse transcriptasepolymerase chain reaction. There were 2 WNV-positive pools of Cx. pipiens/restuans in 2006 and 1 in 2007. Using a bias-corrected maximum likelihood estimation, the WNV infection rate for Cx. pipiens/restuans was 5.48/1,000 mosquitoes in 2006 and 1.08/1,000 mosquitoes in 2007. Gravid Cx. pipiens or Cx. restuans were tested individually for avian Plasmodium by a restriction enzymebased assay. Twelve mosquitoes were positive for avian Plasmodium (10.0), 2 were positive for Haemoproteus, and 3 were positive for Leucocytozoon. There were 4 mixed infections, with mosquitoes positive for >1 of the hemosporidian parasites. This work documents a high rate of hemosporidian infection in Culex spp. and illustrates the potential for co-infections with other arboviruses in bird-feeding mosquitoes and their avian hosts. In addition, hemosporidian infection rates may be a useful tool for investigating the ecological dynamics of Culex/avian interactions. ?? 2010 by The American Mosquito Control Association, Inc.

  16. Living with avian FLU⬝Persistence of the H5N1 highly pathogenic avian influenza virus in Egypt.

    Science.gov (United States)

    Njabo, Kevin Yana; Zanontian, Linda; Sheta, Basma N; Samy, Ahmed; Galal, Shereen; Schoenberg, Frederic Paik; Smith, Thomas B

    2016-05-01

    H5N1 highly pathogenic avian influenza virus (HPAIV) continues to cause mortality in poultry and threaten human health at a panzootic scale in Egypt since it was reported in 2006. While the early focus has been in Asia, recent evidence suggests that Egypt is an emerging epicenter for the disease. Despite control measures, epizootic transmission of the disease continues. Here, we investigate the persistence of HPAIV across wild passerine birds and domestic poultry between 2009 and 2012 and the potential risk for continuous viral transmission in Egypt. We use a new weighted cross J-function to investigate the degree and spatial temporal nature of the clustering between sightings of infected birds of different types, and the risk of infection associated with direct contact with infected birds. While we found no infection in wild birds, outbreaks occurred year round between 2009 and 2012, with a positive interaction between chickens and ducks. The disease was more present in the years 2010 and 2011 coinciding with the political unrest in the country. Egypt thus continues to experience endemic outbreaks of avian influenza HPAIV in poultry and an increased potential risk of infection to other species including humans. With the current trends, the elimination of the HPAIV infection is highly unlikely without a complete revamp of current policies. The application of spatial statistics techniques to these types of data may help us to understand the characteristics of the disease and may subsequently allow practitioners to explore possible preventive solutions. PMID:27066713

  17. Phylogenetic and Pathogenic Analyses of Avian Influenza A H5N1 Viruses Isolated from Poultry in Vietnam

    OpenAIRE

    Zhao, Dongming; Liang, Libin; Li, Yanbing; Jiang, Yongping; Liu, Liling; Chen, Hualan

    2012-01-01

    Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public d...

  18. Low prevalence of avian influenza virus in shorebirds on the Pacific coast of North America

    Science.gov (United States)

    Iverson, Samuel A.; Takekawa, John Y.; Schwarzbach, Steven; Cardona, Carol J.; Warnock, Nils; Bishop, Mary Anne; Schirato, Greg A.; Paroulek, Sara; Ackerman, Joshua T.; Ip, Hon; Boyce, Walter M.

    2008-01-01

    The emergence of highly pathogenic avian influenza (HPAI) H5N1 has elevated concerns about wild birds as virus hosts; however, little is known about the ecological and epidemiological factors of transmission by shorebirds. Here we summarize results for 2,773 shorebirds that were live-trapped on the Pacific coast of the United States during 2006-2007 and tested for avian influenza virus using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation. As was the case throughout North America, HPAI H5N1 was not detected in shorebirds during this interval. Contrary to other wild bird groups, most notably waterfowl, the prevalence of even low pathogenicity virus among shorebirds in our study areas in California, Washington, and Alaska was extremely low (0.5%). Virus was detected by RT-PCR from four different species, including, Dunlin (Calidris alpina; N = 3), Western Sandpiper (C. mauri; N = 8), Long-billed Dowitcher (Limnodromus scolopaceus; N = 1), and American Avocet (Recurvirostra americana; N = 1), with the detections in the latter three constituting the first published records for these birds. Based on studies in the eastern United States, we expected, but did not detect (H1 = 1.6, P = 0.21) elevated avian influenza prevalence among shorebirds during spring migration. Diagnostic tests, which were designed to evaluate testing and sampling methods, indicated poor functioning of traditional virus isolation methods and no improvement in detection likelihood by collecting oropharyngeal swabs in addition to cloacal swab samples for low pathogenicity viruses (Z1 = 0.7, P = 0.48).

  19. Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells.

    Science.gov (United States)

    Qian, Kun; Gao, Ai-jun; Zhu, Ming-yue; Shao, Hong-xia; Jin, Wen-jie; Ye, Jian-qiang; Qin, Ai-jian

    2014-11-01

    To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design. PMID:25197039

  20. Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction--positive

    Science.gov (United States)

    Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embr...

  1. Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus.

    OpenAIRE

    Kung, H J; Fung, Y. K.; Majors, J E; Bishop, J M; Varmus, H E

    1981-01-01

    The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DN...

  2. Estimation of transmission parameters of H5N1 avian influenza virus in chickens.

    Directory of Open Access Journals (Sweden)

    Annemarie Bouma

    2009-01-01

    Full Text Available Despite considerable research efforts, little is yet known about key epidemiological parameters of H5N1 highly pathogenic influenza viruses in their avian hosts. Here we show how these parameters can be estimated using a limited number of birds in experimental transmission studies. Our quantitative estimates, based on Bayesian methods of inference, reveal that (i the period of latency of H5N1 influenza virus in unvaccinated chickens is short (mean: 0.24 days; 95% credible interval: 0.099-0.48 days; (ii the infectious period of H5N1 virus in unvaccinated chickens is approximately 2 days (mean: 2.1 days; 95%CI: 1.8-2.3 days; (iii the reproduction number of H5N1 virus in unvaccinated chickens need not be high (mean: 1.6; 95%CI: 0.90-2.5, although the virus is expected to spread rapidly because it has a short generation interval in unvaccinated chickens (mean: 1.3 days; 95%CI: 1.0-1.5 days; and (iv vaccination with genetically and antigenically distant H5N2 vaccines can effectively halt transmission. Simulations based on the estimated parameters indicate that herd immunity may be obtained if at least 80% of chickens in a flock are vaccinated. We discuss the implications for the control of H5N1 avian influenza virus in areas where it is endemic.

  3. Preparation of Anti-Idiotypic Antibody against Avian Influenza Virus Subtype H9

    Institute of Scientific and Technical Information of China (English)

    BaoquanLi; JunPeng; ZhongxiangNiu; XunheYin; FaxiaoLiu

    2005-01-01

    To generate monoclonal anti-idiotypic antibodies (mAb2) against avian influenza virus subtype H9 (H9 AIV), BALB/c mice were immunized with purified chicken anti-H9-AIV IgG and the splenocytes of immunized mice were fused with myeloma cells NS-1. Hybridoma cells were screened by indirect enzyme-linked immunosorbent assays with both chicken and rabbit anti-H9-AIV IgG as coating antigens. One hybridoma cell clone secreting monoclonal antibody against idiotypes shared by both chicken and rabbit anti-H9-AIV IgG was established. Experiments demonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AIV IgG and to induce chickens to generate hemagglutination inhibition antibodies, indicating this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus. Cellular & Molecular Immunology. 2005;2(2):155-157.

  4. Preparation of Anti-Idiotypic Antibody against Avian Influenza Virus Subtype H9

    Institute of Scientific and Technical Information of China (English)

    Baoquan Li; Jun Peng; Zhongxiang Niu; Xunhe Yin; Faxiao Liu

    2005-01-01

    To generate monoclonal anti-idiotypic antibodies (mAb2) against avian influenza virus subtype H9 (H9 AIV),BALB/c mice were immunized with purified chicken anti-H9-AIV IgG and the splenocytes of immunized mice were fused with myeloma cells NS-1. Hybridoma cells were screened by indirect enzyme-linked immunosorbent assays with both chicken and rabbit anti-H9-AIV IgG as coating antigens. One hybridoma cell clone secreting monoclonal antibody against idiotypes shared by both chicken and rabbit anti-H9-AIV IgG was established. Experiments demonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AIV IgG and to induce chickens to generate hemagglutination inhibition antibodies, indicating this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus. Cellular & Molecular Immunology. 2005;2(2):155-157.

  5. Avian influenza virus infection in apparently healthy domestic birds in Sokoto, Nigeria

    Directory of Open Access Journals (Sweden)

    Innocent Okwundu Nwankwo

    2012-09-01

    Full Text Available The study was conducted among apparently healthy birds brought from different local government areas, neighbouring states and across international boundaries to the Sokoto central live bird market between October 2008 and March 2009. Tracheal and cloacal swabs were collected from 221 apparently healthy birds comprising 182 chickens, 3 turkeys, 11 guineafowl, 17 ducks and 8 pigeons. These samples were analysed using nested polymerase chain reaction (nPCR to check for the presence of avian influenza virus. An overall prevalence of 1.4% (3 positive cases was detected with two cases observed in chickens and one in a pigeon. The findings indicate the circulation of avian influenza in the study area. This raises concern for human and animal health due to zoonotic and economic implications of this virus.

  6. Avian sarcoma and leukosis virus gag gene in the Anser anser domesticus genome.

    Science.gov (United States)

    Zhu, F; Jie, H; Lian, L; Qu, L J; Hou, Z C; Zheng, J X; Chen, S Y; Yang, N; Liu, Y P

    2015-01-01

    Endogenous retroviruses are regarded as ideal genetic markers for evolutionary analyses. Birds were some of the initial vertebrates found to contain endogenous retroviruses. However, few studies have investigated the presence and distribution of endogenous retroviruses in goose. In this study, we detected the avian sarcoma and leukosis virus gag gene in the genomic DNA of 8 Chinese native breeds using polymerase chain reaction method. The results indicated that a 1.2-kb avian sarcoma and leukosis virus gag sequence was integrated into all 8 goose breeds. The mean genetic pairwise distance was 0.918% among the investigated geese. To the best of our knowledge, this is the first report demonstrating the presence of the endogenous retroviruses in the domestic goose genome. The genetic structure should be further examined in the domestic goose. PMID:26600497

  7. CLONING AND EXPRESSION OF ENVELOPE GENE OF SUBGROUP J AVIAN LEUKOSIS VIRUS

    Science.gov (United States)

    Avian leukosis virus subgroup J (ALV-J)was identified in the l990's, and causes mye1ocytic myeloid leukosis in meat-type chicken. The envelope (env)gene of ADOL-4817 strain of ALV-J was amplified by po1ymerase chain reaction (PCR)and cloned into TA vector. The size of env gene is about 1.7 kb. A tr...

  8. Detergent inhibited, heat labile nucleoside triphosphatase in cores of avian myeloblastosis virus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis. This t...... triphosphatase probably originates from the viral membranes. In the cores the enzyme is completely inactivated by low concentrations (0.02%) of Nonident P-40. Also, the enzyme is very thermolabile and denatures rapidly at 38 degrees C....

  9. Psittacid Herpesvirus 1 and Infectious Laryngotracheitis Virus: Comparative Genome Sequence Analysis of Two Avian Alphaherpesviruses

    OpenAIRE

    Thureen, Dean R.; Keeler, Calvin L.

    2006-01-01

    Psittacid herpesvirus 1 (PsHV-1) is the causative agent of Pacheco's disease, an acute, highly contagious, and potentially lethal respiratory herpesvirus infection in psittacine birds, while infectious laryngotracheitis virus (ILTV) is a highly contagious and economically significant avian herpesvirus which is responsible for an acute respiratory disease limited to galliform birds. The complete genome sequence of PsHV-1 has been determined and compared to the ILTV sequence, assembled from pub...

  10. Characterization of tumour virus proteins. I. radioimmunoassay of the P27 protein of avian viruses

    International Nuclear Information System (INIS)

    The major structural protein of avian oncornaviruses, a core component of about 27000 daltons, has been measured by radioimmunoassay. The purified protein was labelled with 125Iodine by chloramine-T method. The immune serum titer was defined as the highest serum dilution able to precipitate 50% of the labelled antigon present in the system. Standard competition curve was constructed in order to determine the equivalents of protein, in a system with limiting antibody concentration. In the experimental conditions used, 0.14 ng of AMV-P27 inhibited 50% of 125I-AMV-P27 (1.0 ng) precipitation. The 125I-AMV-P27 vs anti-AMV-P27 system was used to study the competition of normal cells, purified virus suspension, productive cells and supernatant fluids. Most of the chicken ombryo fibroblast showed expression of this viral component. The phenomena of cell transformation, the increase in total protein, and the expression of P27 were studied in rapid transformation of CEF by RSV-SR sub(A)

  11. Protection of poultry against the 2012 Mexican H7N3 highly pathogenic avian influenza virus with inactivated H7 avian influenza vaccines

    Science.gov (United States)

    In June of 2012, an outbreak of highly pathogenic avian influenza (HPAI) H7N3 was reported poultry in Jalisco, Mexico. Since that time the virus has spread to the surrounding States of Guanajuato and Aguascalientes and new outbreaks continue to be reported. To date more than 25 million birds have di...

  12. Avian influenza A viruses: From zoonosis to pandemic

    NARCIS (Netherlands)

    M. Richard (Mathilde); M.T. de Graaf (Marieke); S. Herfst (Sander)

    2014-01-01

    textabstractZoonotic influenza A viruses originating from the animal reservoir pose a threat for humans, as they have the ability to trigger pandemics upon adaptation to and invasion of an immunologically naive population. Of particular concern are the H5N1 viruses that continue to circulate in poul

  13. Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus

    Directory of Open Access Journals (Sweden)

    Marino Michael P

    2010-01-01

    Full Text Available Abstract Background The ability to efficiently and selectively target gene delivery vectors to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. We pursued two different strategies to target lentiviral vector delivery to specific cell types. In one of the strategies, vector particles bearing a membrane-bound stem cell factor sequence plus a separate fusion protein based either on Sindbis virus strain TR339 glycoproteins or the vesicular stomatitis virus G glycoprotein were used to selectively transduce cells expressing the corresponding stem cell factor receptor (c-kit. An alternative approach involved soluble avian sarcoma/leukosis virus receptors fused to cell-specific ligands including stem cell factor and erythropoietin for targeting lentiviral vectors pseudotyped with avian sarcoma/leukosis virus envelope proteins to cells that express the corresponding receptors. Results The titers of unconcentrated vector particles bearing Sindbis virus strain TR339 or vesicular stomatitis virus G fusion proteins plus stem cell factor in the context of c-kit expressing cells were up to 3.2 × 105 transducing units per ml while vector particles lacking the stem cell factor ligand displayed titers that were approximately 80 fold lower. On cells that lacked the c-kit receptor, the titers of stem cell factor-containing vectors were approximately 40 times lower compared to c-kit-expressing cells. Lentiviral vectors pseudotyped with avian sarcoma/leukosis virus subgroup A or B envelope proteins and bearing bi-functional bridge proteins encoding erythropoietin or stem cell factor fused to the soluble extracellular domains of the avian sarcoma/leukosis virus subgroup A or B receptors resulted in efficient transduction of erythropoietin receptor or c-kit-expressing cells. Transduction of erythropoietin receptor-expressing cells mediated by bi-functional bridge proteins was found to be dependent on the dose, the

  14. Psittacid Herpesvirus 1 and Infectious Laryngotracheitis Virus: Comparative Genome Sequence Analysis of Two Avian Alphaherpesviruses

    Science.gov (United States)

    Thureen, Dean R.; Keeler, Calvin L.

    2006-01-01

    Psittacid herpesvirus 1 (PsHV-1) is the causative agent of Pacheco's disease, an acute, highly contagious, and potentially lethal respiratory herpesvirus infection in psittacine birds, while infectious laryngotracheitis virus (ILTV) is a highly contagious and economically significant avian herpesvirus which is responsible for an acute respiratory disease limited to galliform birds. The complete genome sequence of PsHV-1 has been determined and compared to the ILTV sequence, assembled from published data. The PsHV-1 and ILTV genomes exhibit similar structural characteristics and are 163,025 bp and 148,665 bp in length, respectively. The PsHV-1 genome contains 73 predicted open reading frames (ORFs), while the ILTV genome contains 77 predicted ORFs. Both genomes contain an inversion in the unique long region similar to that observed in pseudorabies virus. PsHV-1 is closely related to ILTV, and it is proposed that it be assigned to the Iltovirus genus. These two avian herpesviruses represent a phylogenetically unique clade of alphaherpesviruses that are distinct from the Marek's disease-like viruses (Mardivirus). The determination of the complete genomic nucleotide sequences of PsHV-1 and ILTV provides a tool for further comparative and functional analysis of this unique class of avian alphaherpesviruses. PMID:16873243

  15. Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expression in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A series of adeno-associated viral vectors conraining a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338Awere obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338Awas prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFIXR338A viral stocks on a large scale and higher fiter was established,which can be used for industrial purpose. The titer of rAAV-hFIXR338A was more than 1.25x1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IXknock-out mice were transfected with the rAAV-hFIXR338Ain vitro and in vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng@ (106cells)-1 @ (24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76±64.36) ng@ (106 cells)-1@ (24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFIX.``

  16. Prevalence of antibodies to type A influenza virus in wild avian species using two serologic assays

    Science.gov (United States)

    Brown, Justin D.; Luttrell, M. Page; Berghaus, Roy D.; Kistler, Whitney; Keeler, Shamus P.; Howey, Andrea; Wilcox, Benjamin; Hall, Jeffrey; Niles, Larry; Dey, Amanda; Knutsen, Gregory; Fritz, Kristen; Stallknecht, David E.

    2010-01-01

    Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

  17. Genetic Analysis of Avian Influenza Virus from Wild Birds and Mallards Reared for Shooting in Denmark

    DEFF Research Database (Denmark)

    Handberg, Kurt; Therkildsen, O. R.; Jørgensen, Poul Henrik

    2010-01-01

    Denmark forms a geographical bottleneck along the migration route of many water birds breeding from northeastern Canada to north Siberia that gather to winter in Europe and Africa. Potentially, the concentration of such large numbers of water birds enhances the risk of avian influenza virus (AIV...... the HP H5N1 virus genome showed that it was not related to the LPAIV isolated previously, but closely related to the HPAIV H5 (Asian type) detected in the rest of Europe at that time. Even though only partial sequences were applied, this gave the idea for future full-length sequence studies....

  18. Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus

    OpenAIRE

    Goutama Michael; Murtini Sri; Soejoedono Retno D; He Fang; Kwang Jimmy

    2010-01-01

    Abstract Background Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. Results In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189A...

  19. [Molecular-genetic analysis of the field isolates of avian leucosis viruses in the Russian Federation].

    Science.gov (United States)

    Plotnikov, V A; Grebennikova, T V; Iuzhakov, A G; Dudnikova, E K; Norkina, S N; Zaberezhnyĭ, A D; Aliper, T I; Fadly, A M

    2012-01-01

    Results of monitoring of different subtypes of avian leukosis virus (ALV) from commercial poultry farms in 14 regions of Russian Federation were discussed. Only three regions were found to be negative. ALV was detected in other 11 regions in 46-64% cases (for different regions). The phylogenetic analysis of the genomes for the 12 field isolates of ALV was carried out in different regions of Russian Federation. The isolates belong to different subtypes of the virus and form two large groups. The genomic differences between Russian and foreign isolates within each group range from 5% to 10%. PMID:23248858

  20. New reassortant H5N8 highly pathogenic avian influenza virus from waterfowl in Southern China

    OpenAIRE

    Song, Yafen; Cui, Jin; Song, Hui; Ye, Jiaqi; Zhao, Zhishan; Wu, Siyu; XU, CHENGGANG; Jiao, Peirong; Liao, Ming

    2015-01-01

    New reassortant H5N8 highly pathogenic avian influenza viruses were isolated from waterfowl in Southern China. Blast analysis demonstrated that the PB2 gene in these viruses were most closely related to A/wild duck/Shangdong/628/2011 (H5N1), while their NP genes were both more closely related to A/wild duck/Shandong/1/2011 (H5N1) and A/duck/Jiangsu/k1203/2010 (H5N8). However, the HA, NA, PB1, PA, M, and NS genes had the highest identity with A/duck/Jiangsu/k1203/2010 (H5N8). Phylogenetic anal...

  1. H5N1 avian influenza virus: human cases reported in southern China.

    OpenAIRE

    Crofts, J; Paget, J; Karcher, F.

    2003-01-01

    Two cases of confirmed influenza due to the avian influenza A H5N1 virus were reported last week in Hong Kong (1). The cases occurred in a Hong Kong family who had recently visited Fujian province in southern China. The daughter, aged 8 years, died following a respiratory illness. The cause of her death is unknown. The father and son also had respiratory illnesses; the father died and the son recovered. Both were infected with the H5N1 virus. The mother also had a respiratory illness, which i...

  2. Phylogenetic Analysis of H7N9 Avian Influenza Virus Based on a Novel Mathematical Descriptor

    OpenAIRE

    Yusheng Bai; Tingting Ma; Yuhua Yao; Qi Dai; Ping-an He

    2014-01-01

    A new mathematical descriptor was proposed based on 3D graphical representation. Using the method, we construct the phylogenetic trees of nine proteins of H7N9 influenza virus to analyze the originated source of H7N9. The results show that the evolution route of H7N9 avian influenza is from America through Europe to Asia. Furthermore, two samples collected from environment in Nanjing and Zhejiang and one sample collected from chicken are the sources of H7N9 influenza virus that infected human...

  3. Dogs are highly susceptible to H5N1 avian influenza virus

    OpenAIRE

    Chen, Ying; Zhong, Gongxun; Wang, Guojun; Deng, Guohua; Li, Yanbing; Shi, Jianzhong; Zhang, Zhuo; Guan, Yuntao; Jiang, Yongping; Bu, Zhigao; Kawaoka, Yoshihiro; Chen, Hualan

    2010-01-01

    Replication of avian influenza viruses (AIVs) in dogs may facilitate their adaptation in humans; however, the data to date on H5N1 influenza virus infection in dogs are conflicting. To elucidate the susceptibility of dogs to this pathogen, we infected two groups of 6 beagles with 106 50% egg-infectious dose of H5N1 AIV A/bar-headed goose/Qinghai/3/05 (BHG/QH/3/05) intranasally (i.n.) and intratracheally (i.t.), respectively. The dogs showed disease symptoms, including anorexic, fever, conjunc...

  4. Chicken interferon alpha pretreatment reduces virus replication of pandemic H1N1 and H5N9 avian influenza viruses in lung cell cultures from different avian species

    Science.gov (United States)

    Type I interferons, including interferon (IFN)-alpha, represent one of the first lines of innate immune defense against influenza virus infection. Following natural infection of chickens with avian influenza virus (AIV), transcription of IFN-alpha is quickly up regulated along with multiple other im...

  5. Newcastle Disease and Avian Influenza A Virus in Migratory Birds in Wetland of Boushehr-Iran

    Directory of Open Access Journals (Sweden)

    M.J. Mehrabanpour

    2011-08-01

    Full Text Available Wild birds are considered to be the natural reservoir of Newcastle Disease Virus (NDV and Avian Influenza virus (AI and are often suspected to be involved in outbreaks in domesticated birds. The objective of the present study was to determine ND and AI infection in migratory birds in the south of Iran in order to detect the possible source of these viruses to domestic poultry. A total of 443 fecal specimens (fresh dropping and cloacal swabs were collected from migratory and wild resident birds in the Bushehr wetlands from October 2009 to June 2010. AI virus was isolated from 3 out of 443 samples processed for virus isolation and confirmed by reverse transcriptase chain reaction (RT-PCR. NDVs were isolated from 22 (fresh fecal samples and were identified as avian paramyxomyxovirus-1 by the results obtained from the HI test with NDV-specific antibodies and RT-PCR-method. Mortality related to NDV was reported in some chicken flocks in the south of Iran. These results, as well as other data from the literature indicate that wild birds play a minor role as a potential disseminator of NDVs and AIVS. This study is the first report of NDV and AIV isolation from migratory and resident birds in the wetlands of Boushehr-Iran. In addition, our findings support the notion that wild aquatic and migratory birds may function as a reservoir for AIV and NDV in the south of Iran.

  6. Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

    Directory of Open Access Journals (Sweden)

    Abdel-Ghany Ahmad E

    2010-04-01

    Full Text Available Abstract Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE. Reverse transcriptase polymerase chain reaction (RT-PCR and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas.

  7. Avian influenza A viruses: from zoonosis to pandemic

    OpenAIRE

    Richard, Mathilde; de Graaf, Miranda; Herfst, Sander

    2014-01-01

    Zoonotic influenza A viruses originating from the animal reservoir pose a threat for humans, as they have the ability to trigger pandemics upon adaptation to and invasion of an immunologically naive population. Of particular concern are the H5N1 viruses that continue to circulate in poultry in numerous countries in Europe, Asia and Africa, and the recently emerged H7N9 viruses in China, due to their relatively high number of human fatalities and pandemic potential. To start a pandemic, zoonot...

  8. Novel reassortant highly pathogenic H5N2 avian influenza viruses in poultry in China.

    Directory of Open Access Journals (Sweden)

    Guo Zhao

    Full Text Available There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11 and A/goose/Eastern China/1112/2011 (GS/EC/1112/11 were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10 and A/duck/Hebei/0908/2009 (DK/HB/0908/09, possess hemagglutinin (HA gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.

  9. A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

    Science.gov (United States)

    Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

    2016-01-01

    Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production. PMID:27222839

  10. Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries.

    Science.gov (United States)

    Körbelin, Jakob; Sieber, Timo; Michelfelder, Stefan; Lunding, Lars; Spies, Elmar; Hunger, Agnes; Alawi, Malik; Rapti, Kleopatra; Indenbirken, Daniela; Müller, Oliver J; Pasqualini, Renata; Arap, Wadih; Kleinschmidt, Jürgen A; Trepel, Martin

    2016-06-01

    Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine. PMID:27018516

  11. Recombinant Newcastle disease virus expressing H9 HA protects chickens against heterologous avian influenza H9N2 virus challenge.

    Science.gov (United States)

    Nagy, Abdou; Lee, Jinhwa; Mena, Ignacio; Henningson, Jamie; Li, Yuhao; Ma, Jingjiao; Duff, Michael; Li, Yonghai; Lang, Yuekun; Yang, Jianmei; Abdallah, Fatma; Richt, Juergen; Ali, Ahmed; García-Sastre, Adolfo; Ma, Wenjun

    2016-05-17

    In order to produce an efficient poultry H9 avian influenza vaccine that provides cross-protection against multiple H9 lineages, two Newcastle disease virus (NDV) LaSota vaccine strain recombinant viruses were generated using reverse genetics. The recombinant NDV-H9Con virus expresses a consensus-H9 hemagglutinin (HA) that is designed based on available H9N2 sequences from Chinese and Middle Eastern isolates. The recombinant NDV-H9Chi virus expresses a chimeric-H9 HA in which the H9 ectodomain of A/Guinea Fowl/Hong Kong/WF10/99 was fused with the cytoplasmic and transmembrane domain of the fusion protein (F) of NDV. Both recombinant viruses expressed the inserted HA stably and grew to high titers. An efficacy study in chickens showed that both recombinant viruses were able to provide protection against challenge with a heterologous H9N2 virus. In contrast to the NDV-H9Chi virus, the NDV-H9Con virus induced a higher hemagglutination inhibition titer against both NDV and H9 viruses in immunized birds, and efficiently inhibited virus shedding through the respiratory route. Moreover, sera collected from birds immunized with either NDV-H9Con or NDV-H9Chi were able to cross-neutralize two different lineages of H9N2 viruses, indicating that NDV-H9Con and NDV-H9Chi are promising vaccine candidates that could provide cross-protection among different H9N2 lineage viruses. PMID:27102817

  12. Novel avian influenza A (H7N9 virus induces impaired interferon responses in human dendritic cells.

    Directory of Open Access Journals (Sweden)

    Veera Arilahti

    Full Text Available In March 2013 a new avian influenza A(H7N9 virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs. We observed that in H7N9 virus-infected cells, interferon (IFN responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced "cytokine storm" seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs.

  13. Hampered foraging and migratory performance in swans infected with low-pathogenic avian influenza A virus.

    Directory of Open Access Journals (Sweden)

    Jan A van Gils

    Full Text Available It is increasingly acknowledged that migratory birds, notably waterfowl, play a critical role in the maintenance and spread of influenza A viruses. In order to elucidate the epidemiology of influenza A viruses in their natural hosts, a better understanding of the pathological effects in these hosts is required. Here we report on the feeding and migratory performance of wild migratory Bewick's swans (Cygnus columbianus bewickii Yarrell naturally infected with low-pathogenic avian influenza (LPAI A viruses of subtypes H6N2 and H6N8. Using information on geolocation data collected from Global Positioning Systems fitted to neck-collars, we show that infected swans experienced delayed migration, leaving their wintering site more than a month after uninfected animals. This was correlated with infected birds travelling shorter distances and fuelling and feeding at reduced rates. The data suggest that LPAI virus infections in wild migratory birds may have higher clinical and ecological impacts than previously recognised.

  14. Characterization of avian sarcoma and leukosis virus receptors

    Czech Academy of Sciences Publication Activity Database

    Elleder, Daniel; Melder, D. C.; Stepanets, Volodymyr; Plachý, Jiří; Geryk, Josef; Federspiel, M. J.; Svoboda, Jan

    Heidelberg : EMBL, 2004 - (Krijnse-Locker, J.; Sodeik, B.; Suomalainen, M.). s. 1 [EMBO Workshop on the Cell Biology of Virus Infection. 25.09.2004-29.09.2004, Heidelberg] Keywords : ASLV * retrovirus receptor Subject RIV: EB - Genetics ; Molecular Biology

  15. Characterization of Avian Influenza and Newcastle Disease Viruses from Poultry in Libya.

    Science.gov (United States)

    Kammon, Abdulwahab; Heidari, Alireza; Dayhum, Abdunaser; Eldaghayes, Ibrahim; Sharif, Monier; Monne, Isabela; Cattoli, Giovanni; Asheg, Abdulatif; Farhat, Milad; Kraim, Elforjani

    2015-09-01

    On March 2013, the Libyan poultry industry faced severe outbreaks due to mixed infections of APMV-1 (Newcastle disease) and low pathogenic avian influenza (AI) of the H9N2 subtype which were causing high mortality and great economic losses. APMV-1 and H9N2 were isolated and characterized. Genetic sequencing of the APMV-1/chicken/Libya/13VIR/ 7225-1/2013 isolate revealed the presence of a velogenic APMV-1 belonging to lineage 5 (GRRRQKR*F Lin.5) or genotype VII in class II, according to the nomenclature in use. Three AI viruses of the H9N2 subtype, namely A/avian/Libya/13VIR7225-2/2013, A/avian/Libya/13VIR7225-3/2013, and A/avian/Libya/13VIR7225-5/2013, were isolated and found to belong to the G1 lineage. Analysis of amino acid sequences showed that the analyzed H9N2 viruses contained the amino acid Leu at position 226 (H3 numbering) at the receptor binding site of the HA, responsible for human virus-like receptor specificity. On March 2014, an outbreak of highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was diagnosed in a backyard poultry farm in an eastern region of Libya. The H5N1 isolate (A/chicken/Libya/14VIR2749-16/2014) was detected by real time RT-PCR (rRT-PCR). Genetic characterization of the HA gene revealed that the identified subtype was highly pathogenic, belonged to the 2.2.1 lineage, and clustered with recent Egyptian viruses. This study revealed the presence of a velogenic APMV-1 genotype and of two influenza subtypes, namely HPAI H5N1 and H9N2, which are of major interest for public and animal health. Considering these findings, more investigations must be undertaken to establish and implement adequate influenza surveillance programs; this would allow better study of the epidemiology of APMV-1 genotype VII in Libya and evaluation of the current vaccination strategies. PMID:26478162

  16. Ecology, Evolution and Pathogenesis of Avian Influenza Viruses

    OpenAIRE

    Munster, Vincent

    2006-01-01

    textabstractInfluenza A virus behoort tot de familie van Orthomyxoviridae. Infl uenza A virussen zijn onregelmatig gevormde virussen van ongeveer 120 nm groot. Het genoom van influenza A virussen is gesegmenteerd en bestaat uit negatief-strengs RNA. De acht gensegmenten coderen voor 11 verschillende eiwitten. Infl uenza A virussen worden onderverdeeld op basis van de oppervlakte eiwitten; hemagglutinine (HA, een eiwit dat zorg draagt voor de binding van het virus aan en binnendringen van de g...

  17. Avian influenza virus (H5N1; effects of physico-chemical factors on its survival

    Directory of Open Access Journals (Sweden)

    Hameed Sajid

    2009-03-01

    Full Text Available Abstract Present study was performed to determine the effects of physical and chemical agents on infective potential of highly pathogenic avian influenza (HPAI H5N1 (local strain virus recently isolated in Pakistan during 2006 outbreak. H5N1 virus having titer 108.3 ELD50/ml was mixed with sterilized peptone water to get final dilution of 4HA units and then exposed to physical (temperature, pH and ultraviolet light and chemical (formalin, phenol crystals, iodine crystals, CID 20, virkon®-S, zeptin 10%, KEPCIDE 300, KEPCIDE 400, lifebuoy, surf excel and caustic soda agents. Harvested amnio-allantoic fluid (AAF from embryonated chicken eggs inoculated with H5N1 treated virus (0.2 ml/egg was subjected to haemagglutination (HA and haemagglutination inhibition (HI tests. H5N1 virus lost infectivity after 30 min at 56°C, after 1 day at 28°C but remained viable for more than 100 days at 4°C. Acidic pH (1, 3 and basic pH (11, 13 were virucidal after 6 h contact time; however virus retained infectivity at pH 5 (18 h, 7 and 9 (more than 24 h. UV light was proved ineffectual in inactivating virus completely even after 60 min. Soap (lifebuoy®, detergent (surf excel® and alkali (caustic soda destroyed infectivity after 5 min at 0.1, 0.2 and 0.3% dilution. All commercially available disinfectants inactivated virus at recommended concentrations. Results of present study would be helpful in implementing bio-security measures at farms/hatcheries levels in the wake of avian influenza virus (AIV outbreak.

  18. Distribution of sialic acid receptors and influenza A viruses of avian and swine origin and in experimentally infected pigs

    DEFF Research Database (Denmark)

    Trebbien, Ramona; Larsen, Lars Erik; Viuff, Birgitte M.

    2011-01-01

    Background: Pigs are considered susceptible to influenza A virus infections from different host origins because earlier studies have shown that they have receptors for both avian (sialic acid-alpha-2,3-terminal saccharides (SAalpha- 2,3)) and swine/human (SA-alpha-2,6) influenza viruses in the...

  19. Subgroup J Avian Leukosis Virus Neutralizing Antibody Escape Variants Contribute to Viral Persistence in Meat-Type Chickens

    Science.gov (United States)

    We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (NAb) against the inoculated parental virus (V+A+) in commercial meat-type chickens inoculated at hatch with Subgroup J avian leukosis virus (ALV J) field isolates. I...

  20. Use of molecularly cloned avian leukosis virus to study antigenic variation following infection of meat-type chickens

    Science.gov (United States)

    A molecularly cloned strain of subgroup J avian leukosis virus (ALV-J) termed R5-4 was used to study antigenic variation following infection of meat-type chickens. Chickens were inoculated with R5-4 virus at either 8 days of embryonation or at 1 week of age. Each chicken was housed in a separate is...

  1. Pathogenicity of recombinant H5N1 avian influenza viruses with truncated NS1 gene in chickens

    Science.gov (United States)

    The NS1 protein of influenza A virus plays an important role in blocking the induction of type I interferon and other regulatory functions in infected cells. However, differences in length of the NS1 protein has been observed in highly pathogenic H5N1, H5N2, and H7N1 subtype avian influenza viruses...

  2. Improved avian influenza virus isolation rates from wild waterfowl cloacal swabs using yolk sac inoculation of embryonating chicken egg

    Science.gov (United States)

    Avian influenza virus (AIV) remains of interest to researchers as a pathogen that infects many economically important bird species. Asymptomatic wild birds, such as waterfowl species, can shed virus and spread it to domestic poultry, where it can cause severe damage. Effective laboratory methods t...

  3. Fatal H5N6 Avian Influenza Virus Infection in a Domestic Cat and Wild Birds in China

    OpenAIRE

    Zhijun Yu; Xiaolong Gao; Tiecheng Wang; Yanbing Li; Yongcheng Li; Yu Xu; Dong Chu; Heting Sun; Changjiang Wu; Shengnan Li; Haijun Wang; Yuanguo Li; Zhiping Xia; Weishi Lin; Jun Qian

    2015-01-01

    H5N6 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H5N6 virus infection in 2014. Here, we report the first cases of fatal H5N6 avian influenza virus (AIV) infection in a domestic cat and wild birds. These cases followed human H5N6 infections in China and preceded an H5N6 outbreak in chickens. The extensive migration routes of wild birds may contribute to the geographic spread of H5N6 AIVs and pose a risk to humans ...

  4. Characterization of an Avian Influenza Virus H9N2 Strain Isolated from a Wild Bird in Southern China

    OpenAIRE

    Xu, Qian; Xie, Zhixun; Xie, Liji; Xie, Zhiqin; Deng, Xianwen; Liu, Jiabo; Luo, Sisi

    2014-01-01

    We isolated an avian influenza virus H9N2 strain from a wild bird in the Guangxi Province of southern China in 2013 named A/turtledove/Guangxi/49B6/2013(H9N2) (GX49B6). We aimed to understand the genetic characters of the GX49B6 strain by analyzing the complete genome sequence. The results showed that our isolated strain has features of low pathogenic avian influenza viruses and viruses that infect humans. The discovery of the complete genome sequence of the GX49B6 strain may be helpful to fu...

  5. Label-free microcantilever-based immunosensors for highly sensitive determination of avian influenza virus H9

    International Nuclear Information System (INIS)

    We report on label-free immunosensors for the highly sensitive detection of avian influenza virus. The method makes use of the microcantilevers of an atomic force microscope onto which monoclonal antibodies against avian influenza virus were covalently immobilized. The factors influencing the performance of the resulting immunosensors were optimized by measuring the deflections of the cantilever via optical reflection, and this resulted in low detection limits and a wide analytical range. The differential deflection signals revealed specific antigen binding and their intensity is proportional to the logarithm of the concentrations of the virus in solution. Under optimal conditions, the immunosensors exhibit a linear response in the 7.6 ng mL−1 to 76 μg mL−1 concentration range of avian influenza virus, and the detection limit is 1.9 ng mL−1. (author)

  6. The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

    OpenAIRE

    Earp, Laurie J.; Delos, Sue E.; Netter, Robert C.; Bates, Paul; White, Judith M.

    2003-01-01

    We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral...

  7. Genetically Diverse Low Pathogenicity Avian Influenza A Virus Subtypes Co-Circulate among Poultry in Bangladesh

    Science.gov (United States)

    Gerloff, Nancy A.; Khan, Salah Uddin; Zanders, Natosha; Balish, Amanda; Haider, Najmul; Islam, Ausraful; Chowdhury, Sukanta; Rahman, Mahmudur Ziaur; Haque, Ainul; Hosseini, Parviez; Gurley, Emily S.; Luby, Stephen P.; Wentworth, David E.; Donis, Ruben O.; Sturm-Ramirez, Katharine; Davis, C. Todd

    2016-01-01

    Influenza virus surveillance, poultry outbreak investigations and genomic sequencing were assessed to understand the ecology and evolution of low pathogenicity avian influenza (LPAI) A viruses in Bangladesh from 2007 to 2013. We analyzed 506 avian specimens collected from poultry in live bird markets and backyard flocks to identify influenza A viruses. Virus isolation-positive specimens (n = 50) were subtyped and their coding-complete genomes were sequenced. The most frequently identified subtypes among LPAI isolates were H9N2, H11N3, H4N6, and H1N1. Less frequently detected subtypes included H1N3, H2N4, H3N2, H3N6, H3N8, H4N2, H5N2, H6N1, H6N7, and H7N9. Gene sequences were compared to publicly available sequences using phylogenetic inference approaches. Among the 14 subtypes identified, the majority of viral gene segments were most closely related to poultry or wild bird viruses commonly found in Southeast Asia, Europe, and/or northern Africa. LPAI subtypes were distributed over several geographic locations in Bangladesh, and surface and internal protein gene segments clustered phylogenetically with a diverse number of viral subtypes suggesting extensive reassortment among these LPAI viruses. H9N2 subtype viruses differed from other LPAI subtypes because genes from these viruses consistently clustered together, indicating this subtype is enzootic in Bangladesh. The H9N2 strains identified in Bangladesh were phylogenetically and antigenically related to previous human-derived H9N2 viruses detected in Bangladesh representing a potential source for human infection. In contrast, the circulating LPAI H5N2 and H7N9 viruses were both phylogenetically and antigenically unrelated to H5 viruses identified previously in humans in Bangladesh and H7N9 strains isolated from humans in China. In Bangladesh, domestic poultry sold in live bird markets carried a wide range of LPAI virus subtypes and a high diversity of genotypes. These findings, combined with the seven year

  8. Genetically Diverse Low Pathogenicity Avian Influenza A Virus Subtypes Co-Circulate among Poultry in Bangladesh.

    Directory of Open Access Journals (Sweden)

    Nancy A Gerloff

    Full Text Available Influenza virus surveillance, poultry outbreak investigations and genomic sequencing were assessed to understand the ecology and evolution of low pathogenicity avian influenza (LPAI A viruses in Bangladesh from 2007 to 2013. We analyzed 506 avian specimens collected from poultry in live bird markets and backyard flocks to identify influenza A viruses. Virus isolation-positive specimens (n = 50 were subtyped and their coding-complete genomes were sequenced. The most frequently identified subtypes among LPAI isolates were H9N2, H11N3, H4N6, and H1N1. Less frequently detected subtypes included H1N3, H2N4, H3N2, H3N6, H3N8, H4N2, H5N2, H6N1, H6N7, and H7N9. Gene sequences were compared to publicly available sequences using phylogenetic inference approaches. Among the 14 subtypes identified, the majority of viral gene segments were most closely related to poultry or wild bird viruses commonly found in Southeast Asia, Europe, and/or northern Africa. LPAI subtypes were distributed over several geographic locations in Bangladesh, and surface and internal protein gene segments clustered phylogenetically with a diverse number of viral subtypes suggesting extensive reassortment among these LPAI viruses. H9N2 subtype viruses differed from other LPAI subtypes because genes from these viruses consistently clustered together, indicating this subtype is enzootic in Bangladesh. The H9N2 strains identified in Bangladesh were phylogenetically and antigenically related to previous human-derived H9N2 viruses detected in Bangladesh representing a potential source for human infection. In contrast, the circulating LPAI H5N2 and H7N9 viruses were both phylogenetically and antigenically unrelated to H5 viruses identified previously in humans in Bangladesh and H7N9 strains isolated from humans in China. In Bangladesh, domestic poultry sold in live bird markets carried a wide range of LPAI virus subtypes and a high diversity of genotypes. These findings, combined with the

  9. Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression Cassette

    OpenAIRE

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; WANG, HONGLI; Xiao, Weidong

    2008-01-01

    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FV...

  10. Updated Values for Molecular Diagnosis for Highly Pathogenic Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Akira Sakurai

    2012-08-01

    Full Text Available Highly pathogenic avian influenza (HPAI viruses of the H5N1 strain pose a pandemic threat. H5N1 strain virus is extremely lethal and contagious for poultry. Even though mortality is 59% in infected humans, these viruses do not spread efficiently between humans. In 1997, an outbreak of H5N1 strain with human cases occurred in Hong Kong. This event highlighted the need for rapid identification and subtyping of influenza A viruses (IAV, not only to facilitate surveillance of the pandemic potential of avian IAV, but also to improve the control and treatment of infected patients. Molecular diagnosis has played a key role in the detection and typing of IAV in recent years, spurred by rapid advances in technologies for detection and characterization of viral RNAs and proteins. Such technologies, which include immunochromatography, quantitative real-time PCR, super high-speed real-time PCR, and isothermal DNA amplification, are expected to contribute to faster and easier diagnosis and typing of IAV.

  11. Genomic and Phylogenetic Characterization of Novel, Recombinant H5N2 Avian Influenza Virus Strains Isolated from Vaccinated Chickens with Clinical Symptoms in China

    OpenAIRE

    Huaiying Xu; Fang Meng; Dihai Huang; Xiaodan Sheng; Youling Wang; Wei Zhang; Weishan Chang; Leyi Wang; Zhuoming Qin

    2015-01-01

    Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses d...

  12. EMERGENCE OF SUBGROUP J AVIAN LEUKOSIS VIRUS NEUTRALIZING ANTIBODY ESCAPE VARIANTS IN MEAT-TYPE CHICKENS INFECTED WITH VIRUS AT HATCH

    Science.gov (United States)

    Infection of meat-type chickens at hatch with field isolates of Subgroup J avian leukosis virus (ALV J) results in a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (NAb) against the inoculated parental virus (V+A+). The purpose of this study was t...

  13. EFFECTS OF VIRULENT AND VACCINE STRAINS OF MAREK'S DISEASE VIRUS ON SUBGROUP J AVIAN LEUKOSIS VIRUS INFECTION IN MEAT-TYPE CHICKENS

    Science.gov (United States)

    The objective of this study was to determine the influence of virulent and vaccine strains of Marek's disease virus (MDV) on subgroup J avian leukosis virus (ALV-J) -induced viremia and cloacal shedding in meat-type chickens. Chickens from two lines were infected with ALV-J at hatch; chickens were ...

  14. Pathogenesis of the novel avian-origin influenza A (H7N9) virus Influenza H7N9 virus in human lower respiratory tract

    OpenAIRE

    Chan, LY; Chan, WY; Peiris, JSM; Chan, MCW

    2013-01-01

    Background: As of May 2013, 131 laboratory-confirmed human infections with a novel influenza H7N9 virus had been reported from China. The source of human infection appears to be poultry. There is so far no evidence of sustained human-to-human transmission. Genetic analysis revealed that all eight gene segments of H7N9 were of avian origin; six internal gene segments from avian influenza H7N9 viruses, while hemagglutinin and neuraminidase genes were derived from influenza viruses c...

  15. Spatial assessment of the potential risk of avian influenza A virus infection in three raptor species in Japan

    Science.gov (United States)

    MORIGUCHI, Sachiko; ONUMA, Manabu; GOKA, Koichi

    2016-01-01

    Avian influenza A, a highly pathogenic avian influenza, is a lethal infection in certain species of wild birds, including some endangered species. Raptors are susceptible to avian influenza, and spatial risk assessment of such species may be valuable for conservation planning. We used the maximum entropy approach to generate potential distribution models of three raptor species from presence-only data for the mountain hawk-eagle Nisaetus nipalensis, northern goshawk Accipiter gentilis and peregrine falcon Falco peregrinus, surveyed during the winter from 1996 to 2001. These potential distribution maps for raptors were superimposed on avian influenza A risk maps of Japan, created from data on incidence of the virus in wild birds throughout Japan from October 2010 to March 2011. The avian influenza A risk map for the mountain hawk-eagle showed that most regions of Japan had a low risk for avian influenza A. In contrast, the maps for the northern goshawk and peregrine falcon showed that their high-risk areas were distributed on the plains along the Sea of Japan and Pacific coast. We recommend enhanced surveillance for each raptor species in high-risk areas and immediate establishment of inspection systems. At the same time, ecological risk assessments that determine factors, such as the composition of prey species, and differential sensitivity of avian influenza A virus between bird species should provide multifaceted insights into the total risk assessment of endangered species. PMID:26972333

  16. Antibodies to H5 subtype avian influenza virus and Japanese encephalitis virus in northern pintails (Anas acuta) sampled in Japan

    Science.gov (United States)

    Ramey, Andy M.; Spackman, Erica; Yeh, Jung-Yong; Fujita, Go; Konishi, Kan; Reed, John A.; Wilcox, Benjamin R.; Brown, Justin D.; Stallknecht, David E.

    2013-01-01

    Blood samples from 105 northern pintails (Anas acuta) captured on Hokkaido, Japan were tested for antibodies to avian influenza virus (AIV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) to assess possible involvement of this species in the spread of economically important and potentially zoonotic pathogens. Antibodies to AIV were detected in 64 of 105 samples (61%). Of the 64 positives, 95% and 81% inhibited agglutination of two different H5 AIV antigens (H5N1 and H5N9), respectively. Antibodies to JEV and WNV were detected in five (5%) and none of the samples, respectively. Results provide evidence for prior exposure of migrating northern pintails to H5 AIV which couldhave implications for viral shedding and disease occurrence. Results also provide evidence for limited involvement of this species in the transmission and spread of flaviviruses during spring migration.

  17. Duplex PCR assay for the detection of avian adeno virus and chicken anemia virus prevalent in Pakistan

    Directory of Open Access Journals (Sweden)

    Iqbal Aqib

    2011-09-01

    Full Text Available Abstract Avian Adeno viruses and Chicken Anemia Viruses cause serious economic losses to the poultry industry of Pakistan each year. Timely and efficient diagnosis of the viruses is needed in order to practice prevention and control strategies. In the first part of this study, we investigated broilers, breeder and Layer stocks for morbidity and mortality rates due to AAV and CAV infections and any co-infections by examining signs and symptoms typical of their infestation or post mortem examination. In the second part of the study, we developed a duplex PCR assay for the detection of AAV and CAV which is capable to simultaneously detect both the viral types prevalent in Pakistan with high sensitivity and 100% specificity.

  18. Avian influenza virus RNA extraction from tissue and swab material

    Science.gov (United States)

    Viral RNA extraction is a critical step for any molecular downstream application and correct sample collection and handling is critical to obtaining reliable results for virus recovery or detection. The choice of Specimen type depends on numerous factors and must be compatible with the downstream ap...

  19. Evaluation of Nobuto filter paper strips for the detection of avian influenza virus antibody in waterfowl

    Science.gov (United States)

    Dusek, Robert J.; Hall, Jeffrey S.; Nashold, Sean W.; TeSlaa, Joshua L.; Ip, Hon S.

    2011-01-01

    The utility of using Nobuto paper strips for the detection of avian influenza antibodies was examined in mallards (Anas platyrhynchos) experimentally infected with low pathogenic avian influenza viruses. Blood was collected 2 wk after infection and was preserved either as serum or whole blood absorbed onto Nobuto strips. Analysis of samples using a commercially available blocking enzyme-linked immunosorbent assay revealed comparable results (≥96% sensitivity for all methods) between sera stored at -30 C and the Nobuto strip preservation method even when the Nobuto strips were stored up to 3 mo at room temperature (RT). Significant differences were detected in the ratio of sample absorbance to negative control absorbance for Nobuto strips stored at RT compared with sera stored at -30 C, although these differences did not affect the ability of the test to reliably detect positive and negative samples. Nobuto strips are a convenient and sensitive alternative to the collection of serum samples when maintaining appropriate storage temperatures is difficult.

  20. Synergistic Effect of S224P and N383D Substitutions in the PA of H5N1 Avian Influenza Virus Contributes to Mammalian Adaptation

    Science.gov (United States)

    Song, Jiasheng; Xu, Jing; Shi, Jianzhong; Li, Yanbing; Chen, Hualan

    2015-01-01

    The adaptation of H5N1 avian influenza viruses to human poses a great threat to public health. Previous studies indicate the adaptive mutations in viral polymerase of avian influenza viruses are major contributors in overcoming the host species barrier, with the majority of mammalian adaptive mutations occurring in the PB2 protein. However, the adaptive mutations in the PA protein of the H5N1 avian influenza virus are less defined and poorly understood. In this study, we identified the synergistic effect of the PA/224P + 383D of H5N1 avian influenza viruses and its ability to enhance the pathogenicity and viral replication in a mammalian mouse model. Interestingly, the signature of PA/224P + 383D mainly exists in mammalian isolates of the H5N1 influenza virus and pdmH1N1 influenza virus, providing a potential pathway for the natural adaptation to mammals which imply the effects of natural adaptation to mammals. Notably, the mutation of PA/383D, which is highly conserved in avian influenza viruses, increases the polymerase activity in both avian and human cells, and may have roles in maintaining the avian influenza virus in their avian reservoirs, and jumping species to infect humans. PMID:26000865

  1. 人感染禽流感病毒的传播%The spread of human infection with avian influenza virus

    Institute of Scientific and Technical Information of China (English)

    陈帅帅; 郭潮潭

    2013-01-01

    Avian influenza virus belongs to type A influenza virus,its infection lead to infectious disease that spread among the avian.During 1997,some avian influenza viruses that present in poultry have across the species barrier,so that it can transmit from avian to humans directly.It has caused the death of many infections in Asia and the whole world,and became a potential pandemic factor.Therefore,the situation of avian influenza infection in humans from 1997 are aualyzed in this review,in order to provide science basis for the prevention and control about the outbreak of new avian influenza in the future.%禽流感病毒属于A型流感病毒,其感染导致的传染病一般只在禽类间传播,然而1997年以来,存在于家禽中的一些禽流感病毒已经突破了动物种间屏障,能够直接从禽类传播给人类,导致亚洲及全球范围内很多感染病例的死亡,存在潜在大流行的威胁.此文对1997年以来禽流感病毒感染人类的状况进行分析,为今后新型禽流感暴发的预防和控制提供参考.

  2. Avian influenza virus ecology in Iceland shorebirds: intercontinental reassortment and movement

    Science.gov (United States)

    Hall, Jeffrey S.; Hallgrimsson, Gunnar Thor; Suwannanarn, Kamol; Sreevatsen, Srinand; Ip, Hon S.; TeSlaa, Joshua L.; Nashold, Sean W.; Dusek, Robert J.

    2014-01-01

    Shorebirds are a primary reservoir of avian influenza viruses (AIV). We conducted surveillance studies in Iceland shorebird populations for 3 years, documenting high serological evidence of AIV exposure in shorebirds, primarily in Ruddy Turnstones (Arenaria interpres; seroprevalence = 75%). However, little evidence of virus infection was found in these shorebird populations and only two turnstone AIVs (H2N7; H5N1) were able to be phylogenetically examined. These analyses showed that viruses from Iceland shorebirds were primarily derived from Eurasian lineage viruses, yet the H2 hemagglutinin gene segment was from a North American lineage previously detected in a gull from Iceland the previous year. The H5N1 virus was determined to be low pathogenic, however the PB2 gene was closely related to the PB2 from highly pathogenic H5N1 isolates from China. Multiple lines of evidence suggest that the turnstones were infected with at least one of these AIV while in Iceland and confirm Iceland as an important location where AIV from different continents interact and reassort, creating new virus genomes. Mounting data warrant continued surveillance for AIV in wild birds in the North Atlantic, including Canada, Greenland, and the northeast USA to determine the risks of new AI viruses and their intercontinental movement in this region.

  3. New Reassortant H5N6 Highly Pathogenic Avian Influenza Viruses in Southern China, 2014

    Science.gov (United States)

    Jiao, Peirong; Cui, Jin; Song, Yafen; Song, Hui; Zhao, Zhishan; Wu, Siyu; Qu, Nannan; Wang, Nianchen; Ouyang, Guowen; Liao, Ming

    2016-01-01

    New reassortant H5N6 highly pathogenic avian influenza viruses (AIVs) were isolated from apparently healthy domestic ducks in Southern China in 2014. Our results show that the viruses grew efficiently in eggs and replicated systemically in chickens. They were completely lethal in chicken (100% mortality), and the mean death time was 6 to 7 days post-inoculation. The viruses could transmit in chickens by naïve contact. BLAST analysis revealed that their HA gene was most closely related to A/wild duck/Shangdong/628/2011 (H5N1), and their NA genes were most closely related to A/swine/Guangdong/K6/2010 (H6N6). The other genes had the highest identity with A/wild duck/Fujian/1/2011(H5N1). The results of phylogenetic analysis showed that their HA genes clustered into clade 2.3.4.4 of the H5N1 viruses and all genes derived from H5 were Mix-like or H6-like viruses. Thus, the new H5N6 viruses were reassortmented of H5N1 and H6N6 virus. Therefore, the circulation of the new H5N6 AIVs may become a threat to poultry and human health. PMID:27242767

  4. The antigenic property of the H5N1 avian influenza viruses isolated in central China

    Directory of Open Access Journals (Sweden)

    Zou Wei

    2012-08-01

    Full Text Available Abstract Background Three influenza pandemics outbroke in the last century accompanied the viral antigen shift and drift, resulting in the change of antigenic property and the low cross protective ability of the existed antibody to the newly emerged pandemic virus, and eventually the death of millions of people. The antigenic characterizations of the viruses isolated in central China in 2004 and 2006–2007 were investigated in the present study. Results Hemagglutinin inhibition assay and neutralization assay displayed differential antigenic characteristics of the viruses isolated in central China in two periods (2004 and 2006–2007. HA genes of the viruses mainly located in two branches in phylogeny analysis. 53 mutations of the deduced amino acids of the HA genes were divided into 4 patterns. Mutations in pattern 2 and 3 showed the main difference between viruses isolated in 2004 and 2006–2007. Meanwhile, most amino acids in pattern 2 and 3 located in the globular head of the HA protein, and some of the mutations evenly distributed at the epitope sites. Conclusions The study demonstrated that a major antigenic drift had occurred in the viruses isolated in central China. And monitoring the antigenic property should be the priority in preventing the potential pandemic of H5N1 avian influenza virus.

  5. Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

    OpenAIRE

    Liang, Libin; Deng, Guohua; Shi, Jianzhong; Wang, Shuai; Zhang, Qianyi; Kong, Huihui; Gu, Chunyang; Guan, Yuntao; Suzuki, Yasuo; Li, Yanbing; Jiang, Yongping; Tian, Guobin; Liu, Liling; Li, Chengjun; Chen, Hualan

    2016-01-01

    ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that...

  6. Lack of evidence that avian oncogenic viruses are infectious for humans: a review.

    Science.gov (United States)

    Schat, Karel A; Erb, Hollis N

    2014-09-01

    Chickens may be infected with three different oncogenic viruses: avian leukosis virus (ALV), reticuloendotheliosis virus (REV), and Marek's disease herpesvirus (MDV). Several epidemiological studies have suggested a link between these viruses and different types of cancer in people working in poultry processing plants and with multiple sclerosis. In this article, we analyze the epidemiological evidence that these viruses are causative agents for human cancer, followed by description of the relevant key characteristics of ALV, REV, and MDV. Finally, we discuss the biological evidence or lack thereof that avian tumor viruses are involved in the etiology of human cancer and multiple sclerosis (MS). The recent primary epidemiologic articles that we reviewed as examples were only hypothesis-generating studies examining massive numbers of risk factors for associations with various imprecise, non-viral-specific outcomes. The studies lacked precise evidence of exposure to the relevant viruses and the statistical methods failed to adjust for the large risks of false-positive claims. ALV subgroups A-D and J have been eradicated in the United States from the pure lines down to the parent stocks by the breeder companies, which have greatly reduced the incidence of infection in layer flocks and broilers. As a consequence, potential exposure of humans to these viruses has greatly diminished. Infection of humans working in processing plants with ALV-A and ALV-B is unlikely, because broilers are generally resistant to infection with these two subgroups. Moreover, these viruses enter cells by specific receptors present on chicken, but not on mammalian, cells. Infection of mammalian cell cultures or animals with ALV-A, ALV-B, and ALV-J has not been reported. Moreover, humans vaccinated with exogenous or endogenous ALV-contaminated vaccines against yellow fever, measles, and mumps did not become antibody- or virus-positive for ALV. The risks for human infection with REV are similarly

  7. Seroepizootiological investigations of animals from Obedska bara locality for presence of Avian influenza virus

    Directory of Open Access Journals (Sweden)

    Đuričić Bosiljka

    2010-01-01

    Full Text Available The disease caused by Influenza viruses has been well known for a very long time. In the recent period there has been noted an occurrence of pandemics caused by Influenza viruses type A with a high rate of mortality. The ongoing pandemic caused by avian influenza virus serotype H9N9 began in Hong Kong in 1992, and another pandemic caused by serotype H5N1 began in China (Hong Kong in 1999. The world wide spreading of these viruses occurred due to migratory birds. Avian influenza was confirmed in Serbia in 2007. The goal of this study was to examine whether the avian influenza viruses type A circulate in the region of the Obedska bara marsh, which is a famous resort for many birds in Serbia, as well as many birds migrating from Europe to Africa and vice versa. The samples of blood sera of many animal species (123 samples from fowl, 64 samples from donkeys, 40 samples from horses were tested by serologic reaction of inhibition of haemmaglutination (IHA for the presence of antibodies to influenza A subtypes H5N1, H5N2, H5N3, H7N1 and H7N2. Also, the samples of blood sera of experimental chicken exposed to wild life in Obedska bara (sentinel species were tested. Antibodies to subtypes H5N1, H5N2, H5N3, H7N1 and H7N2 were found in chicken from Dec, Boljevci, Petrovcic and Kupinovo villages but no antibodies were found in blood sera from hams from Dobanovci, Jakovo, Becmen and Surcin villages. From 23 samples from ducks antibodies were detected in 3 samples, and from 22 geese blood sera antibodies were found in 4 samples. From a total of 40 horse blood sera tested one was tested positive, and from 64 donkey sera 17 were positive for the presence of antibodies for avian influenza type A. In blood sera of experimental chicken antibodies were found by subtype H5N1 with corrections with H5N2 and H7N1.

  8. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail

    Science.gov (United States)

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Hung, Vu-Khac; Nguyen, Duc Tan; Kim, Wonyong

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010) and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014), were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010) and clade 2.3.2.1c (CVVI-50/2014), which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail. PMID:26900963

  9. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail.

    Directory of Open Access Journals (Sweden)

    Tinh Huu Nguyen

    Full Text Available H5N1 highly pathogenic avian influenza (HPAI viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010 and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014, were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010 and clade 2.3.2.1c (CVVI-50/2014, which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail.

  10. Natural Infection with Avian Hepatitis E Virus and Marek's Disease Virus in Brown Layer Chickens in China.

    Science.gov (United States)

    Yang, Shuqing; Wang, Liyuan; Sun, Shuhong

    2016-09-01

    In the present study, avian hepatitis E virus (HEV) and serotype-1 strains of Marek's disease virus (MDV-1) were detected from a flock of 27-wk-old brown layer hens in China, accompanied by an average daily mortality of 0.44%. Postmortem examination of 25 sick hens and five apparently healthy hens selected randomly from the flock showed significant pathologic changes consistent with hepatitis-splenomegaly syndrome (HSS), including hepatomegaly, peritoneal fluid, and hepatic subcapsular hemorrhages. Microscopic examination of these livers showed multifocal necrotizing hepatitis and mild lymphocytic infiltration. These liver samples were investigated for HEV by reverse-transcription PCR. The overall detection rate of HEV RNA in samples of sick chickens was about 56% (14/25), while in samples from apparently healthy hens, it was 80% (4/5). Sequencing analysis of three 242-base-pair fragments of the helicase gene revealed 95.5% to 97.9% nucleotide identity compared with published avian HEV genotype 3, whereas identities demonstrated only 77.3% to 86.0% similarity when compared with genotypes 1, 2, and 4. Unexpectedly, the MDV meq gene was detected in livers from both apparently healthy chickens (2/5) and sick chickens (12/25) by PCR analysis. The meq gene (396 base pairs) was determined to belong to MDV-1 by further sequencing. The co-infection rate of avian HEV and MDV in this flock was 30% (9/30). This is the first report of dual infection of a nonenvelope RNA virus (HEV) with a herpesvirus (MDV) in chickens in China. PMID:27610734

  11. Prevalence of Antibodies to H9N2 Avian Influenza Virus in Backyard Chickens around Maharlou Lake in Iran

    Directory of Open Access Journals (Sweden)

    Mohammad Mehdi Hadipour*, Gholamhossein Habibi and Amir Vosoughi

    2011-06-01

    Full Text Available Backyard chickens play an important role in the epidemiology of H9N2 avian influenza virus infection. Close contact of backyard chickens with migratory birds, especially with aquatic birds, as well as neighboring poultry farms, may pose the risk of transmitting avian influenza virus, but little is known about the disease status of backyard poultry. A H9N2 avian influenza virus seroprevalence survey was carried out in 500 backyard chickens from villages around Maharlou lake in Iran, using the hemagglutination-inhibition (HI test. The studied backyard chickens had not been previously vaccinated and showed no clinical signs of disease. The overall HI titer and seroprevalence against H9N2 were 7.73 and 81.6%, respectively.

  12. Fluorescence biosensor based on CdTe quantum dots for specific detection of H5N1 avian influenza virus

    International Nuclear Information System (INIS)

    This report highlights the fabrication of fluorescence biosensors based on CdTe quantum dots (QDs) for specific detection of H5N1 avian influenza virus. The core biosensor was composed of (i) the highly luminescent CdTe/CdS QDs, (ii) chromatophores extracted from bacteria Rhodospirillum rubrum, and (iii) the antibody of β-subunit. This core part was linked to the peripheral part of the biosensor via a biotin–streptavidin–biotin bridge and finally connected to the H5N1 antibody to make it ready for detecting H5N1 avian influenza virus. Detailed studies of each constituent were performed showing the image of QDs-labeled chromatophores under optical microscope, proper photoluminescence (PL) spectra of CdTe/CdS QDs, chromatophores and the H5N1 avian influenza viruses. (paper)

  13. Fluorescence biosensor based on CdTe quantum dots for specific detection of H5N1 avian influenza virus

    Science.gov (United States)

    Hoa Nguyen, Thi; Dieu Thuy Ung, Thi; Hien Vu, Thi; Tran, Thi Kim Chi; Quyen Dong, Van; Khang Dinh, Duy; Liem Nguyen, Quang

    2012-09-01

    This report highlights the fabrication of fluorescence biosensors based on CdTe quantum dots (QDs) for specific detection of H5N1 avian influenza virus. The core biosensor was composed of (i) the highly luminescent CdTe/CdS QDs, (ii) chromatophores extracted from bacteria Rhodospirillum rubrum, and (iii) the antibody of β-subunit. This core part was linked to the peripheral part of the biosensor via a biotin–streptavidin–biotin bridge and finally connected to the H5N1 antibody to make it ready for detecting H5N1 avian influenza virus. Detailed studies of each constituent were performed showing the image of QDs-labeled chromatophores under optical microscope, proper photoluminescence (PL) spectra of CdTe/CdS QDs, chromatophores and the H5N1 avian influenza viruses.

  14. Immune Efficacy of a Recombinant Fowlpox Virus Co-Ex-pressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

    Institute of Scientific and Technical Information of China (English)

    QIAO Chuan-ling; JIANG Yong-ping; YU Kang-zhen; TIAN Guo-bin; CHEN Hua-lan

    2004-01-01

    A recombinant fowlpox virus co-expressing Haemagglutinin(HA)and Neuraminidase(NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In this study,to evaluate its ability of protecting chickens against challenge with a lethal dose of highly pathogenic isolates of avian influenza virus,eight-week-old specificpathogenic-free(SPF)chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenic avian influenza virus H5N1 and H7N1 isolate,all chickens vaccinated with recombinant virus were protected,while the chickens vaccinated with the wildtype fowlpox virus or unvaccinated controls experienced 100% mortality respectively following challenge. This complete protection was accompanied by the high levels of specific antibody response to the respectivecomponents of the recombinant virus.

  15. Avian influenza A virus PB2 promotes interferon type I inducing properties of a swine strain in porcine dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Ocana-Macchi, Manuela; Ricklin, Meret E.; Python, Sylvie; Monika, Gsell-Albert [Institute of Virology and Immunoprophylaxis, Mittelhaeusern (Switzerland); Stech, Juergen; Stech, Olga [Friedrich-Loeffler Institut, Greifswald-Insel Riems (Germany); Summerfield, Artur, E-mail: artur.summerfield@ivi.admin.ch [Institute of Virology and Immunoprophylaxis, Mittelhaeusern (Switzerland)

    2012-05-25

    The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-{kappa}B translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.

  16. Avian influenza A virus PB2 promotes interferon type I inducing properties of a swine strain in porcine dendritic cells

    International Nuclear Information System (INIS)

    The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.

  17. The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

    Institute of Scientific and Technical Information of China (English)

    JIANG Yong-ping; YU Kang-zhen; DENG Guo-hua; TIAN Guo-bin; QIAO Chuan-ling; CHEN Hua-lan

    2004-01-01

    The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.

  18. Isolation of avian influenza virus (H9N2 from emu in china

    Directory of Open Access Journals (Sweden)

    Kang Wenhua

    2006-03-01

    Full Text Available Abstract This is the first reported isolation of avian influenza virus (AIV from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic AIV (LPAIV, according to OIE standards. It was named A/Emu/HeNen/14/2004(H9N2(Emu/HN/2004. The HA gene (1683bp was amplified by RT-PCR and it was compared with other animal H9N2 AIV sequences in GenBank, the US National Institutes of Health genetic sequence database. The results suggested that Emu/HN/2004 may have come from an avian influenza virus (H9N2 from Southern China.

  19. Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs

    Directory of Open Access Journals (Sweden)

    Meng Qing-Wen

    2011-12-01

    Full Text Available Abstract Background Avian leukosis virus (ALV is a major infectious disease that impacts the poultry industry worldwide. Despite intensive efforts, no effective vaccine has been developed against ALV because of mutations that lead to resistant forms. Therefore, there is a dire need to develop antiviral agents for the treatment of ALV infections and RNA interference (RNAi is considered an effective antiviral strategy. Results In this study, the avian leukosis virus subgroup J (ALV-J proviral genome, including the gag genes, were treated as targets for RNAi. Four pairs of miRNA sequences were designed and synthesized that targeted different regions of the gag gene. The screened target (i.e., the gag genes was shown to effectively suppress the replication of ALV-J by 19.0-77.3%. To avoid the generation of escape variants during virus infection, expression vectors of multi-target miRNAs were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes. Multi-target miRNAs were shown to play a synergistic role in the inhibition of ALV-J replication, with an inhibition efficiency of viral replication ranging from 85.0-91.2%. Conclusion The strategy of multi-target miRNAs might be an effective method for inhibiting ALV replication and the acquisition of resistant mutations.

  20. Synergistic pathogenic effects of co-infection of subgroup J avian leukosis virus and reticuloendotheliosis virus in broiler chickens.

    Science.gov (United States)

    Dong, Xuan; Zhao, Peng; Chang, Shuang; Ju, Sidi; Li, Yang; Meng, Fanfeng; Sun, Peng; Cui, Zhizhong

    2015-01-01

    To study interactions between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) and the effects of co-infection on pathogenicity of these viruses, 1-day-old broiler chicks were infected with ALV-J, REV or both ALV-J and REV. The results indicated that co-infection of ALV-J and REV induced more growth retardation and higher mortality rate than ALV-J or REV single infection (P < 0.05). Chickens co-infected with ALV-J and REV also showed more severe immunosuppression than those with a single infection. This was manifested by significantly lower bursa of Fabricius and thymus to body weight ratios and lower antibody responses to Newcastle disease virus and H9-avian influenza virus (P < 0.05). Perihepatitis and pericarditis related to severe infection with Escherichia coli were found in many of the dead birds. E. coli was isolated from each case of perihepatitis and pericarditis. The mortality associated with E. coli infection in the co-infection groups was significantly higher than in the other groups (P < 0.05). Among 516 tested E. coli isolates from 58 dead birds, 12 serotypes of the O-antigen were identified in two experiments. Different serotypes of E. coli strains were even isolated from the same organ of the same bird. Diversification of O-serotypes suggested that perihepatitis and pericarditis associated with E. coli infection was the most frequent secondary infection following the immunosuppression induced by ALV-J and REV co-infection. These results suggested that the co-infection of ALV-J and REV caused more serious synergistic pathogenic effects, growth retardation, immunosuppression, and secondary E. coli infection in broiler chickens. PMID:25484188

  1. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  2. Extensive geographic mosaicism in avian influenza viruses from gulls in the northern hemisphere.

    Directory of Open Access Journals (Sweden)

    Michelle Wille

    Full Text Available Due to limited interaction of migratory birds between Eurasia and America, two independent avian influenza virus (AIV gene pools have evolved. There is evidence of low frequency reassortment between these regions, which has major implications in global AIV dynamics. Indeed, all currently circulating lineages of the PB1 and PA segments in North America are of Eurasian origin. Large-scale analyses of intercontinental reassortment have shown that viruses isolated from Charadriiformes (gulls, terns, and shorebirds are the major contributor of these outsider events. To clarify the role of gulls in AIV dynamics, specifically in movement of genes between geographic regions, we have sequenced six gull AIV isolated in Alaska and analyzed these along with 142 other available gull virus sequences. Basic investigations of host species and the locations and times of isolation reveal biases in the available sequence information. Despite these biases, our analyses reveal a high frequency of geographic reassortment in gull viruses isolated in America. This intercontinental gene mixing is not found in the viruses isolated from gulls in Eurasia. This study demonstrates that gulls are important as vectors for geographically reassorted viruses, particularly in America, and that more surveillance effort should be placed on this group of birds.

  3. Efficient Sensing of Avian Influenza Viruses by Porcine Plasmacytoid Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Artur Summerfield

    2011-03-01

    Full Text Available H5N1 influenza A virus (IAV infections in human remain rare events but have been associated with severe disease and a higher mortality rate compared to infections with seasonal strains. An excessive release of pro-inflammatory cytokine together with a greater virus dissemination potential have been proposed to explain the high virulence observed in human and other mammalian and avian species. Among the cells involved in the cytokine storm, plasmacytoid dendritic cells (pDC could play an important role considering their unique capacity to secrete massive amounts of type I interferon (IFN. Considering the role of IFN as a major component of antiviral responses as well as in priming inflammatory responses, we aimed to characterize the induction of IFN-α release upon infection with IAV originating from various avian and mammalian species in a comparative way. In our porcine pDC model, we showed that the viral components triggering IFN responses related to the ability to hemagglutinate, although virosomes devoid of viral RNA were non-stimulatory. Heat-treatment at 65 °C but not chemical inactivation destroyed the ability of IAV to stimulate pDC. All IAV tested induced IFN-α but at different levels and showed different dose-dependencies. H5 and H7 subtypes, in particular H5N1, stimulated pDC at lower doses when compared to mammalian IAV. At high viral doses, IFN-α levels reached by some mammalian IAV surpassed those induced by avian isolates. Although sialic acid-dependent entry was demonstrated, the α-2,3 or α-2,6 binding specificity alone did not explain the differences observed. Furthermore, we were unable to identify a clear role of the hemagglutinin, as the IFN-a doses-response profiles did not clearly differ when viruses with all genes of identical avian origin but different HA were compared. This was found with IAV bearing an HA derived from either a low, a high pathogenic H5N1, or a human H3. Stimulation of pDC was associated with p

  4. Avian influenza A H5N1 virus: a continuous threat to humans

    OpenAIRE

    To, Kelvin KW; Ng, Kenneth HL; Que, Tak-Lun; Chan, Jacky MC; Tsang, Kay-Yan; Tsang, Alan KL; Chen, Honglin; Yuen, Kwok-Yung

    2012-01-01

    We report the first case of severe pneumonia due to co-infection with the emerging avian influenza A (H5N1) virus subclade 2.3.2.1 and Mycoplasma pneumoniae. The patient was a returning traveller who had visited a poultry market in South China. We then review the epidemiology, virology, interspecies barrier limiting poultry-to-human transmission, clinical manifestation, laboratory diagnosis, treatment and control measures of H5N1 clades that can be transmitted to humans. The recent controvers...

  5. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines.

    OpenAIRE

    Stoker, A W; BISSELL, M. J.

    1988-01-01

    We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreo...

  6. Infrequent involvement of c-fos in avian leukosis virus-induced nephroblastoma.

    OpenAIRE

    Collart, K L; Aurigemma, R; Smith, R. E.; Kawai, S; Robinson, H L

    1990-01-01

    To determine whether c-fos is involved in avian leukosis virus-induced nephroblastoma, 28 tumors from chickens were analyzed for novel fos fragments. DNA from 1 of 16 clonal outgrowths (in chicken 6561) contained novel fos-related EcoRI and KpnI fragments which hybridized to both v-fos and viral probes. Oncogenicity tests using filtered 6561 tumor cell homogenates did not reveal a tumor-inducing transduction of c-fos. We conclude that c-fos is only an occasional target for proviral insertions...

  7. Novel sequences of subgroup J avian leukosis viruses associated with hemangioma in Chinese layer hens

    OpenAIRE

    Pan Wei; Gao Yulong; Sun Fenfen; Qin Litin; Liu Zaisi; Yun Bingling; Wang Yongqiang; Qi Xiaole; Gao Honglei; Wang Xiaomei

    2011-01-01

    Abstract Background Avian leukosis virus subgroup J (ALV-J) preferentially induces myeloid leukosis (ML) in meat-type birds. Since 2008, many clinical cases of hemangioma rather than ML have frequently been reported in association with ALV-J infection in Chinese layer flocks. Results Three ALV-J strains associated with hemangioma were isolated and their proviral genomic sequences were determined. The three isolates, JL093-1, SD09DP03 and HLJ09MDJ-1, were 7,670, 7,670, and 7,633 nt in length. ...

  8. Complete Genome Sequence of a J Subgroup Avian Leukosis Virus Isolated from Local Commercial Broilers

    OpenAIRE

    Li, Hongxin; Xue, Chunyi; Ji, Jun; CHANG, SHUANG; Shang, Huiqin; Zhang, Lingjun; Ma, Jingyun; Bi, Yingzuo; Xie, Qingmei

    2012-01-01

    Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5′-GGCACCTCC-3′) for c-myb was identified in the GDKP1202 genome. These findings will provide additional insight...

  9. Surveillance for Avian Influenza Viruses in Wild Birds in Denmark and Greenland, 2007–10

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Breum, Solvej Østergaard; Trebbien, Ramona; Handberg, Kurt Jensen; Therkildsen, Ole Roland; Madsen, Jesper Johannes; Thorup, Kasper; Baroch, John A.; DeLiberto, Thomas J.; Larsen, Lars Erik; Jørgensen, Poul Henrik

    In Denmark and Greenland, extensive surveillance of avian influenza (AI) viruses in wild bird populations has been conducted from 2007 through 2010. In Denmark, the surveillance consisted of passive surveillance of wild birds found dead or sick across Denmark and active surveillance of apparently...... were birds that were found dead. In Greenland, samples were collected mainly from fecal droppings in breeding areas. Samples from 3555 live and apparently healthy wild birds were tested. All swab samples were tested by pan-influenza reverse transcriptase–PCR (RT-PCR), and the positive samples were...

  10. Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

    DEFF Research Database (Denmark)

    De Vleeschauwer, Annebel; Atanasova, Kalina; Van Borm, Steven; van den Berg, Thierry; Rasmussen, Thomas Bruun; Uttenthal, Åse; Van Reeth, Kristien

    2009-01-01

    Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to...... compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a...... only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were...

  11. Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full-Length Nonstructural Gene in Pichia pastoris

    OpenAIRE

    Abubakar, M. B.; I. Aini; Omar, A. R.; Hair-Bejo, M

    2011-01-01

    Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA...

  12. The Genomic Contributions of Avian H1N1 Influenza A Viruses to the Evolution of Mammalian Strains

    OpenAIRE

    Koçer, Zeynep A.; Carter, Robert; Wu, Gang; Zhang, Jinghui; Webster, Robert G.

    2015-01-01

    Among the influenza A viruses (IAVs) in wild aquatic birds, only H1, H2, and H3 subtypes have caused epidemics in humans. H1N1 viruses of avian origin have also caused 3 of 5 pandemics. To understand the reappearance of H1N1 in the context of pandemic emergence, we investigated whether avian H1N1 IAVs have contributed to the evolution of human, swine, and 2009 pandemic H1N1 IAVs. On the basis of phylogenetic analysis, we concluded that the polymerase gene segments (especially PB2 and PA) circ...

  13. Complete Genome Sequence Analysis of an H6N1 Avian Influenza Virus Isolated from Guangxi Pockmark Ducks

    OpenAIRE

    Xie, Zhixun; Xie, Liji; Zhou, Chenyu; Liu, Jiabo; Pang, Yaoshan; Deng, Xianwen; Xie, Zhiqin; Fan, Qing

    2012-01-01

    We report here the complete genomic sequence of a novel H6N1 avian influenza virus strain, A/Duck/Guangxi/GXd-5/2010(H6N1), isolated from pockmark ducks in Guangxi Province, Southern China. All of the 8 gene segments of A/Duck/Guangxi/GXd-5/2010(H6N1) are attributed to the Eurasian lineage; the amino acid motif of the cleavage site between HA1 and HA2 was P-Q-I-E-T-R-G. These are typical characteristics of the low-pathogenicity avian influenza virus. This study will help to understand the epi...

  14. Evolution of Highly Pathogenic H5N1 Avian Influenza Viruses in Vietnam between 2001 and 2007

    OpenAIRE

    Wan, Xiu-Feng; Nguyen, Tung; Davis, C Todd; Smith, Catherine B.; Zhao, Zi-ming; Carrel, Margaret; Inui, Kenjiro; Do, Hoa T.; Mai, Duong T.; Jadhao, Samadhan; Balish, Amanda; Shu, Bo; Luo, Feng; Emch, Michael; Matsuoka, Yumiko

    2008-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic an...

  15. Characterization of Y73, an avian sarcoma virus: a unique transforming gene and its product, a phosphopolyprotein with protein kinase activity.

    OpenAIRE

    Kawai, S; Yoshida, M; Segawa, K; Sugiyama, H; Ishizaki, R; Toyoshima, K

    1980-01-01

    The Y73 strain of avian sarcoma virus recently isolated in Japan is defective in replication and is associated with subgroup A leukosis virus (YAV). The virus caused sarcoma but not acute leukosis when inoculated into chickens. Studies on the viral RNA showed that a 26S RNA, etimated to be 4.8 kilobases long, was Y73 viral RNA carrying a transforming gene. The 26S RNA has sequences in common with the RNA of an avian leukosis virus but no homology with the src gene sequence of avian sarcoma vi...

  16. Field Investigation on the Prevalence of Avian Influenza Virus Infection in Some Localities in Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Abdullah N. Alkhalaf

    2010-07-01

    Full Text Available The objective of this study was to find out prevalence and types of avian influenza virus (AIV among broilers, native chickens, ducks and pigeons in Saudi Arabia. Field investigation was carried out in four localities including Al-Qassim, Hail, Al-Jouf and Northern Border regions. Serum sample, tracheal and cloacal swabs were collected from broilers (n=1561, layers (n=988, ducks (n=329 and pigeons (n=450 from these localities and tested for three different avian influenza viruses (H9, H5 and H3 using Enzyme linked immunosorbent (ELISA test, hamagglutination inhibition (HI test and polymerase chain reaction (PCR. All tested samples were negative for H5 and H3 viruses. In contrast, all positive results were found to be for H9 AI virus using PCR, ELISA and HI test. Chicken sera tested by ELISA for AIV revealed the highest positive samples in Northern Border regions (45.71%, followed by Al-Jouf (29.65%, Al-Qassim (23.98% and Hial (20.94% with non-significant difference (χ2=5.983; P=0.112. HI test carried out on duck sera revealed 35.90% prevalence of antibodies against AIV. PCR amplification resulted in 34.28 and 21.36% positive samples in ducks and chickens, respectively. The highest (45.71% PCR positive chicken samples were from Northern Border regions, followed by Al-Jouf (24.13%, Al-Qassim (19.30% and Hail (16.69% with significant difference (χ2=7.620; P=0.055. All tested pigeons samples were negative for the three virus serotypes included in the study.

  17. Diverse inter-continental and host lineage reassortant avian influenza A viruses in pelagic seabirds.

    Science.gov (United States)

    Huang, Yanyan; Robertson, Gregory J; Ojkic, Davor; Whitney, Hugh; Lang, Andrew S

    2014-03-01

    Avian influenza A viruses (AIVs) often infect waterfowl, gulls and shorebirds, but other bird groups including pelagic seabirds also serve as hosts. In this study, we analyzed 21 AIVs found in two distant breeding colonies of Common Murre (Uria aalge) in Newfoundland and Labrador, Canada, during 2011. Phylogenetic analyses and genotype assignments were performed for the 21 Common Murre viruses together with all Common and Thick-billed Murre (Uria lomvia) AIV sequences available in public sequence databases. All fully characterized viruses from the Common Murres in 2011 were H1N2 subtype, but the genome sequences revealed greater diversity and the viruses belonged to four distinct genotypes. The four genotypes shared most segments in common, but reassortment was observed for PB2 and M segments. This provided direct genetic data of AIV diversification through segment reassortment during an outbreak of AIV infection in high-density breeding colonies. Analysis of the total collection of available murre viruses revealed a diverse collection of subtypes and gene lineages with high similarity to those found in viruses from waterfowl and gulls, and there was no indication of murre-specific AIV gene lineages. Overall, the virus gene pool in murres was predominantly made up of AIV lineages associated with waterfowl, but also featured considerable gull lineage genes and inter-continental reassortments. In particular, all but one of the 21 Common Murre viruses from 2011 in Newfoundland contained 1 or 2 Eurasian segments and 16 contained 1 gull lineage segment. This mosaic nature of characterized murre AIV genomes might reflect an under-recognized role of these pelagic seabirds in virus transmission across space and between bird host taxa. PMID:24462905

  18. Evidence of Avian Leukosis Virus Subgroup E and Endogenous Avian Virus in Marek’s Disease Vaccines Derived from Chicken Embryo Fibroblasts

    Directory of Open Access Journals (Sweden)

    N.R. Dhanutha

    2012-12-01

    Full Text Available The aim of this study was to detect and characterize the endogenous ALVs in cell associated MD vaccine. Chicken embryo fibroblast cell associated Marek’s disease vaccine was tested for possible contamination with Avian Leukosis Viruses (ALVs. Initially the vaccine cell lysate was tested for presence of group specific antigen (p27 of ALVs by ELISA and found positive for GSA. Subsequently total DNA and RNA was isolated from vaccine CEFs and analyzed by PCR and RT-PCR using primers specific for ALV subgroups A-E and J. Subgroup specific PCR and RT-PCR revealed that the CEFs were positive for ALV-E and negative for all other exogenous ALV subgroups (ALV-A, B, C, D and J. Envelope gp85 gene sequence alignment and phylogenetic analysis further confirmed that the ALV sequences found in CEFs of MD vaccine were belongs to endogenous ALV-E. Further this sequence has high homology with endogenous loci ev-1, ev-3 and ev-6. Amplification of genomic DNA with endogenous virus locus specific primers revealed that the CEFs of MD vaccine possess ev-1 and ev-6 and negative for ev-3, ev-9 and ev-21. In conclusion, the data in this study clearly demonstrated that the cell associated commercial MD vaccine tested was contaminated with an endogenous subgroup E and also possess ev-loci such as ev1 and ev-6.

  19. Phylogenetic and pathogenic analyses of avian influenza A H5N1 viruses isolated from poultry in Vietnam.

    Directory of Open Access Journals (Sweden)

    Dongming Zhao

    Full Text Available Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature.

  20. Phylogenetic and Pathogenic Analyses of Avian Influenza A H5N1 Viruses Isolated from Poultry in Vietnam

    Science.gov (United States)

    Li, Yanbing; Jiang, Yongping; Liu, Liling; Chen, Hualan

    2012-01-01

    Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature. PMID:23226433